| 1. |
- Gray, A. C., et al.
(författare)
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Animal-based antibodies : Obsolete
- 2016
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 353:6298, s. 452-453
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Tidskriftsartikel (refereegranskat)
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| 2. |
- Gray, A. C., et al.
(författare)
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Animal-Friendly Affinity Reagents : Replacing the Needless in the Haystack
- 2016
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Ingår i: Trends in Biotechnology. - : Elsevier BV. - 0167-7799. ; 34:12, s. 960-969
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Forskningsöversikt (refereegranskat)abstract
- The multibillion-dollar global antibody industry produces an indispensable resource but that is generated using millions of animals. Despite the irrefutable maturation and availability of animal-friendly affinity reagents (AFAs) employing naïve B lymphocyte or synthetic recombinant technologies expressed by phage display, animal immunisation is still authorised for antibody production. Remarkably, replacement opportunities have been overlooked, despite the enormous potential reduction in animal use. Directive 2010/63/EU requires that animals are not used where alternatives exist. To ensure its implementation, we have engaged in discussions with the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) and the Directorate General for Environment to carve out an EU-led replacement strategy. Measures must be imposed to avoid outsourcing, regulate commercial production, and ensure that antibody producers are fully supported.
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| 3. |
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| 4. |
- Delfani, P., et al.
(författare)
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Deciphering systemic lupus erythematosus-associated serum biomarkers reflecting apoptosis and disease activity
- 2017
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Ingår i: Lupus. - : SAGE Publications. - 0961-2033 .- 1477-0962. ; 26:4, s. 373-387
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Tidskriftsartikel (refereegranskat)abstract
- Systemic lupus erythematosus (SLE) is a severe chronic inflammatory autoimmune connective tissue disease. Despite major efforts, SLE remains a poorly understood disease with unpredictable course, unknown etiology and complex pathogenesis. Apoptosis combined with deficiency in clearing apoptotic cells is an important etiopathogenic event in SLE, which could contribute to the increased load of potential autoantigen(s); however, the lack of disease-specific protein signatures deciphering SLE and the underlying biological processes is striking and represents a key limitation. In this retrospective pilot study, we explored the immune system as a specific sensor for disease, in order to advance our understanding of SLE. To this end, we determined multiplexed serum protein expression profiles of crude SLE serum samples, using antibody microarrays. The aim was to identify differential immunoprofiles, or snapshots of the immune response modulated by the disease, reflecting apoptosis, a key process in the etiology of SLE and disease activity. The results showed that multiplexed panels of SLE-associated serum biomarkers could be decoded, in particular reflecting disease activity, but potentially the apoptosis process as well. While the former biomarkers could display a potential future use for prognosis, the latter biomarkers might help shed further light on the apoptosis process taking place in SLE.
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| 5. |
- Dueñas, M., et al.
(författare)
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In vitro immunization of naive human B cells yields high affinity immunuglobulin G antibodies as illustrated by phage display
- 1996
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Ingår i: Immunology. - 0019-2805. ; 89:1, s. 1-7
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Tidskriftsartikel (refereegranskat)abstract
- In vitro antibody responses to a synthetic immunogen, consisting of both a B cell [V3 loop of gp120 from human immunodeficiency virus type 1 (HIV-1)] and a T-helper epitope (15 amino acids of tetanus toxoid) was studied. The in vitro activation was performed by primary and secondary in vitro immunizations, using lymphocytes obtained from uninfected, seronegative donors. Analysis of the in vitro immune response demonstrated an antigen-specific isotype switch, which was dependent on the presence of antigen-specific T-helper cells, CD40 ligation and antigen. Antibody libraries were constructed from cells derived directly from the naive donors, or from primary or secondary in vitro immunized B cells. Five libraries were displayed on filamentous phage and selected for anti-V3-specific Fab fragments, using a selection approach that linked recognition and phage replication. A panel of 19 recombinant antigen-specific Fab, representing different phases of the humoral in vitro immune response, were sequenced, expressed and analysed using a biosensor. Recombinant Fab fragments derived from cultures on day 12 exhibited an increase in affinity of close to two orders of magnitude compared to those obtained from cells primary immunized for 7 days. This study provides the first evidence that an antigen-specific in vitro immune response can yield high-affinity immunoglobuling G antibodies.
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| 6. |
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| 7. |
- Mellby, Linda D., et al.
(författare)
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Serum biomarker signature-based liquid biopsy for diagnosis of early-stage pancreatic cancer
- 2018
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Ingår i: Journal of Clinical Oncology. - 0732-183X. ; 36:28, s. 2887-2894
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Tidskriftsartikel (refereegranskat)abstract
- Purpose Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis, with a 5-year survival of, 10% because of diffuse symptoms leading to late-stage diagnosis. That survival could increase significantly if localized tumors could be detected early. Therefore, we used multiparametric analysis of blood samples to obtain a novel biomarker signature of early-stage PDAC. The signature was derived from a large patient cohort, including patients with well-defined early-stage (I and II) PDAC. This biomarker signature was validated subsequently in an independent patient cohort. Patients and Methods The biomarker signature was derived from a case-control study, using a Scandinavian cohort, consisting of 16 patients with stage I, 132 patients with stage II, 65 patients with stage III, and 230 patients with stage IV PDAC, and 888 controls. This signature was validated subsequently in an independent case-control cohort in the United States with 15 patients with stage I, 75 patients with stage II, 15 patients with stage III, and 38 patients with stage IV PDAC, and 219 controls. An antibody microarray platform was used to identify the serum biomarker signature associated with early-stage PDAC. Results Using the Scandinavian case-control study, a biomarker signature was created, discriminating samples derived from patients with stage I and II from those from controls with a receiver operating characteristic area under the curve value of 0.96. This signature, consisting of 29 biomarkers, was then validated in an independent case-control study in the United States. The biomarker signature could discriminate patients with stage I and II PDAC from controls in this independent patient cohort with a receiver operating characteristic area under the curve value of 0.96. Conclusion This serum biomarker signature might represent a tenable approach to detecting early-stage, localized PDAC if these findings are supported by a prospective validation study.
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| 8. |
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| 9. |
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| 10. |
- Borrebaeck, Carl, et al.
(författare)
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Protein chips based on recombinant antibody fragments: A highly sensitive approach as detected by mass spectrometry
- 2001
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Ingår i: BioTechniques. - 0736-6205. ; 30:5, s. 1126-1126
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Tidskriftsartikel (refereegranskat)abstract
- With the human genome in a first sequence draft and several other genomes being finished this year, the existing information gap between genomics and proteomics is becoming increasingly evident. The analysis of the proteome is, however, much more complicated because the synthesis and structural requirements of functional proteins are different from the easily handled oligonucleotides for which a first analytical breakthrough already has come in the use of DNA chips. In comparison with the DNA microarrays, the protein arrays, or protein chips, offer the distinct possibility of developing a rapid global analysis of the entire proteome. Thus, the concept of comparing proteomic maps of healthy and diseased cells may allow us to understand cell signaling and metabolic pathways and will form a novel base for pharmaceutical companies to develop future therapeutics much more rapidly. This report demonstrates the possibilities of designing protein chips based on specially constructed, small recombinant antibody fragments using nanostructure surfaces with biocompatible characteristics, resulting in sensitive detection in the 600-amol range. The assay readout allows the determination of single or multiple antigen-antibody interactions. Mass identity of the antigens, currently with a resolution of 8000, enables the detection of structural modifications of single proteins.
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| 11. |
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| 12. |
- Broos, Sissela, et al.
(författare)
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Synergistic augmentation of CD40-mediated activation of antigen-presenting cells by amphiphilic poly(γ-glutamic acid) nanoparticles.
- 2012
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Ingår i: Biomaterials. - : Elsevier BV. - 1878-5905 .- 0142-9612. ; 33:26, s. 6230-6239
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Tidskriftsartikel (refereegranskat)abstract
- Agonistic anti-CD40 monoclonal antibodies (mAbs) hold great potential for cancer immunotherapy. However, systemic administration of anti-CD40 mAbs can be associated with severe side effects, such as cytokine release syndrome and liver damage. With the aim to increase the immunostimulatory potency as well as to achieve a local drug retention of anti-CD40 mAbs, we linked an agonistic mAb to immune activating amphiphilic poly(γ-glutamic acid) nanoparticles (γ-PGA NPs). We demonstrate that adsorption of anti-CD40 mAb to γ-PGA NPs (anti-CD40-NPs) improved the stimulatory capacity of the CD40 agonist, resulting in upregulation of costimulatory CD80 and CD86 on antigen-presenting cells, as well as IL-12 secretion. Interestingly, anti-CD40-NPs induced strong synergistic proliferative effects in B cells, possibly resulting from a higher degree of CD40 multimerization, enabled by display of multiple anti-CD40 mAbs on the NPs. In addition, local treatment with anti-CD40-NPs, compared to only soluble CD40 agonist, resulted in a significant reduction in serum levels of IL-6, IL-10, IL-12 and TNF-α in a bladder cancer model. Taken together, our results suggest that anti-CD40-NPs are capable of synergistically enhancing the immunostimulatory effect induced by the CD40 agonist, as well as minimizing adverse side effects associated with systemic cytokine release. This concept of nanomedicine could play an important role in localized immunotherapy of cancer.
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| 13. |
- Chin, L. T., et al.
(författare)
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Site-directed primary in vitro immunization : Production of HIV-1 neutralizing human monoclonal antibodies from lymphocytes obtained from seronegative donors
- 1994
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Ingår i: Immunology. - 0019-2805. ; 81:3, s. 428-434
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Tidskriftsartikel (refereegranskat)abstract
- The design of an in vitro immunization system based on a synthetic heterotope immunogen, which was a peptide containing both T- and B-cell epitopes, that elicited a neutralizing, primary human humoral immune response against the human immunodeficiency virus (HIV-1) is reported here. This heterotope construct contained the major neutralizing B-cell epitope, within the V3 region of glycoprotein 120 (gp120), linked to a promiscuous helper T- cell epitope of tetanus toxin. The peptide was used to induce a human humoral in vitro immune response against the V3 region, using lymphocytes obtained from healthy, sero-negative blood donors. The in vitro immunized peripheral blood lymphocytes were Epstein-Barr virus infected and the antibody response to the synthetic peptide was evaluated using a solid-phase ELISA with the recombinant C-terminal fragment of gp120 (pB1, amino acid residues 287 467, derived from the HIV-1 LAI isolate). The heterotope construct yielded a significant frequency of specifically immunized B cells, in contrast to the control immunizations with individual T and B epitopes mixtures of these epitopes or no immunogen at all. This approach allowed us to generate human monoclonal antibodies, using lymphocytes derived from sero-negative donors, that cross-neutralized several HIV-1 strains, inhibited syncytia formation as well as prevented spreading of the viral infection from cell to cell. Thus, site-directed in vitro immunization using synthetic heterotopes might prove valuable in the dissection and induction of a protective humoral immune response.
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| 14. |
- Cruz, Silian, et al.
(författare)
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Mouse monoclonal antibodies against outer membrane proteins of a vaccine strain of Neisseria meningitidis B : 4:P1.15
- 1998
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Ingår i: Minerva Biotecnologica. - 1120-4826. ; 10:2, s. 65-70
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Tidskriftsartikel (refereegranskat)abstract
- Background. Neisseria meningitidis (Nm) is a Gram negative diplococcus causing bacterial meningitis and fulminant septicemia. In order to allow efficient characterization of infecting strains, antibody reagents for use as analytical tools have proven to be invaluable tools. Similarly, antibodies against relevant bacterial antigens may guide in the selection of components to be included in developing vaccine strategies. Methods. We have thus developed mouse monoclonal antibodies specific for class 1, 3 and 5 antigens expressed by the B:4:P1.15 isolate CU385/83, also being used in a recently developed protective vaccine. In particular, two antibodies CB-Nm.1 and CB- Nm.2 recognize epitopes partly overlapping the subserotype (class 1 antigens) and serotype (class 3 antigen) specificities detected by the previously defined antibodies C6 and 15-1-P4 respectively, were evaluated. Results. As judged by strain recognition, the absolute requirement for binding differs between both the class 1-specific and class 3 specific antibodies suggesting the importance of using multiple antibodies when evaluating subserotype/serotype characteristics of clinical isolates of Nm by serological methods. Conclusion. Furthermore, the development of antibodies crossreactive with subserotype/serotype antigens may partly explain the ability of outer membrane protein vaccine to induce protective activity against strains considered as carrying different class 1 and 3 antigens as determined by available (sub)serotyping reagents.
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| 15. |
- Dueñas, M., et al.
(författare)
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A point mutation in a murine immunoglobulin V-region strongly influences the antibody yield in Escherichia coli
- 1995
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Ingår i: Gene. - 0378-1119. ; 158:1, s. 61-66
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Tidskriftsartikel (refereegranskat)abstract
- Recombinant DNA technology has made it possible to produce specific Fab and scFv antibody (Ab) fragments in prokaryotic host cells. Using vectors designed for periplasmic expression of encoded Ab fragments, we have been studying how the sequence and genetic localization of the light chain (L-chain) variable region gene of a mouse Ab (CB-Nm.1) determined the level of Ab production. The variable region was shown to belong to the VKV family and contained a previously unreported Ile72. Nine different Ab constructions were tested in monocistronic (scFv) or dicistronic (Fab) operons for their ability to affect the synthesis level of the L-chain. When the gene coding for the L-chain was located downstream from the Fd fragment gene, the substitution of codons encoding Ile by a codon encoding Thr was found to be crucial for any expression of the L-chain fragment. This was, however, not accompanied by an increase in L-chain-specific mRNA, neither was there any change in the size of the mRNA. The fact that the unmutated L-chain protein was produced from cells transformed with certain other constructions indicated that the protein as such was not incompatible with the prokaryotic environment. Together, this suggested that the translation process was involved in the restricted production of the L-chain. Thus, surprisingly small substitutions significantly affected the expression level, a fact that will have important implications on the library size expressed in prokaryotic hosts, including phagedisplayed Ab libraries.
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| 16. |
- Duenas, M., et al.
(författare)
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Intra- and extracellular expression of an scFv antibody fragment in E. coli : Effect of bacterial strains and pathway engineering using GroES/L chaperonins
- 1994
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Ingår i: BioTechniques. - 0736-6205. ; 16:3, s. 476-480
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Tidskriftsartikel (refereegranskat)abstract
- We have studied the influence of bacterial host on the secretion of single-chain Fv antibody fragment (scFv), the production of this antibody fragment as intracellular fusion protein, and the effect of chaperonin coexpression on intracellular antibody expression. Seven bacterial strains were transformed with a vector carrying the genes encoding the variable regions of an anti-CEA scFv antibody and the ompA leader sequence (ptrp/ompA/scFvCEA). Expression and secretion of this antibody fragment were highest in the W3110 strain, as determined by Western blot analysis and enzyme immunoassay, where the scFv fragment amounted to approximately 30% of the total periplasmic protein. Except for BMH71-18, the other strains were unsuitable for antibody fragment expression, suggesting screening of bacterial strains as an important parameter. For intracellular expression, the scFv was expressed as a fusion protein with a 26-amino acid N-terminal fragment of human interleukin-2 (IL-2), using the pIL-2f/scFvCEA vector. The fusion protein was expressed at 30% of total biomass and retained antigen binding after in vitro refolding. Co-expression of chaperonin encoding plasmic pGroES/L with pIL-2f/scFv increased the intracellular production of the fusion protein two-fold, with a similar increase in the final amount of active scFv antibody fragment that could be obtained after in vitro refolding. The chaperonins had no effect on secretion of scFv antibody fragments, using the ptrp/ompA/scFvCEA.
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| 17. |
- Dueñas, Marta, et al.
(författare)
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Selection of phage displayed antibodies based on kinetic constants
- 1996
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Ingår i: Molecular Immunology. - : Elsevier BV. - 0161-5890. ; 33:3, s. 279-285
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Tidskriftsartikel (refereegranskat)abstract
- The display of antibody fragments on the surface of filamentous bacteriophages and the selection of binders from antibody libraries have provided powerful tools to generate human antibodies. We reported recently a new concept (SAP system) for the selection of specific phages by linking antigenic recognition and phage replication, using a soluble fusion protein containing the antigen and a fragment of the M13 coat protein 3. In this investigation, a model library has been composed using six different antibody fragments which were characterized individually regarding their k(ass), k(diss) and K(a). All Feb fragments were specific for a 15 amino acid region of the V3 loop of gp120 (HIV-1). We demonstrated that the SAP system could discriminate between the kinetic parameters of each clone, using different selection strategies. Phages expressing high affinity clones were selected preferentially using low doses of antigen but clones of lower affinity also could be selected by increasing the antigen concentration or using a preselection procedure. Phages expressing antibody fragment with high association or low dissociation rate constants were retrieved by utilizing short contact times between antigen and antibody or antigen-chase conditions.
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| 18. |
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| 19. |
- Ifversen, P., et al.
(författare)
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Effect of cell-derived growth factors and cytokines on the clonal outgrowth of EBV-infected B cells and established lymphoblastoid cell lines
- 1993
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Ingår i: Human Antibodies and Hybridomas. - 0956-960X. ; 4:3, s. 115-123
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Tidskriftsartikel (refereegranskat)abstract
- Epstein-Barr virus (EBV) is a potent inducer of polyclonal B lymphocyte proliferation and is widely used as a tool for the establishment of B cell lines producing human monoclonal antibodies. However, because of low transformability, low clonability, and the inherent instability of EBV-infected B cells, valuable antibody-producing B cells are often lost during this procedure. We have here examined various cell-derived cytokines for their ability to enhance both the cellular outgrowth of newly infected B cells and the clonability of infected B cells and lymphoblastoid cell lines. Our results show that the murine thymoma cell line EL-4 is superior to peripheral blood mononuclear cells in both cellular outgrowth and cloning experiments, whereas monocyte-derived factors and monocyte cell lines were less capable than peripheral blood mononuclear cells in enhancing cellular outgrowth and cloning. Furthermore, the human T cell hybridoma cell line MP6 that secretes a B cell growth and differentiation factor, recently identified as an isoform of thioredoxin, is also capable of stimulating EBV-infected B cells and lymphoblastoid cell lines. Co-cultivation of EBV-infected B cells with MP6 cells significantly enhanced the cloning efficiency at the 1 cell/well level. The present results also suggest that one potential role of the MP6-derived thioredoxin could be the up regulation of IL-6 receptor expression in EBV-infected B cells.
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| 20. |
- Jirholt, P., et al.
(författare)
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Exploiting sequence space : Shuffling in vivo formed complementarity determining regions into a master framework
- 1998
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Ingår i: Gene. - 0378-1119. ; 215:2, s. 471-476
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Tidskriftsartikel (refereegranskat)abstract
- A novel approach in molecular design is presented, where in vivo formed complementarity determining regions (CDR) from antibody genes were shuffled into a specific framework region. A synthetic gene library of soluble VH-fragments was created and the complexity of the library was determined by sequencing. The synthetic genes were diverse and contained random combinations of CDR from different germlines. All CDR were randomised in one step and by using in vivo formed CDR, the length, sequence and combination were varied simultaneously.
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| 21. |
- Kuci Emruli, Venera, et al.
(författare)
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Identification of a serum biomarker signature associated with metastatic prostate cancer
- 2021
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Ingår i: Proteomics - Clinical Applications. - : Wiley. - 1862-8346 .- 1862-8354. ; 15:2-3
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Improved early diagnosis and determination of aggressiveness of prostate cancer (PC) is important to select suitable treatment options and to decrease over-treatment. The conventional marker is total prostate specific antigen (PSA) levels in blood, but lacks specificity and ability to accurately discriminate indolent from aggressive disease. Experimental design: In this study, we sought to identify a serum biomarker signature associated with metastatic PC. We measured 157 analytes in 363 serum samples from healthy subjects, patients with non-metastatic PC and patients with metastatic PC, using a recombinant antibody microarray. Results: A signature consisting of 69 proteins differentiating metastatic PC patients from healthy controls was identified. Conclusions and clinical relevance: The clinical value of this biomarker signature requires validation in larger independent patient cohorts before providing a new prospect for detection of metastatic PC.
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| 22. |
- Lindstedt, Malin, et al.
(författare)
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Individuals with occupational allergy to detergent enzymes display a differential transcriptional regulation and cellular immune response
- 2005
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Ingår i: Clinical and Experimental Allergy. - : Wiley. - 0954-7894 .- 1365-2222. ; 35:2, s. 199-206
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Tidskriftsartikel (refereegranskat)abstract
- Background: In spite of significant safety measures, allergy to industrial enzymes remains a major concern. The increasing prevalence of occupational allergy emphasizes the need to investigate the functional properties of enzyme-exposed dendritic cells (DCs), as DCs possess a potent ability to activate allergen-specific T cells. Objective: This study aims at elucidating the molecular mechanisms underlying allergic immune responses to lipase, an industrial enzyme. For this purpose, we studied the effect of both hypoallergenic and wild-type lipase on the transcriptional regulation in DCs and their stimulatory effect on memory CD4+ T cells. Methods: Five individuals with documented lipase allergy were tested for specific serum IgE. DCs from these individuals, stimulated with lipases, were assayed for their ability to affect proliferation and polarization of memory T cells. The effect of lipases on transcriptional activity in DCs was evaluated using global expression analysis. Results: Lipase-specific IgE levels varied considerably between donors, with donor 4 exhibiting highest levels, and a potent specific CD4+ T cell recall response was demonstrated only for donor 4. No difference was detected in cytokine profile when T cells from donor 4 were co-cultured with DCs pulsed with either hypoallergenic or wild-type lipase, as demonstrated by high IL-4 and IL-13, and low IFN-? production. However, the lipases induced different genetic signatures in DCs from donor 4, as compared with the non-responders. Conclusions: DCs from individuals with clinically diagnosed allergy to lipase displayed a differential response to stimulation with hypoallergenic and wild-type lipase in vitro. Only allergen-pulsed DCs from donor 4 were able to induce CD4+ T cell proliferation. The lipase-specific T cells displayed a T-helper type 2 phenotype, which was not altered by hypoallergenic lipase-pulsed DCs. Furthermore, DCs derived from donor 4 and stimulated with either of the lipases displayed different transcriptional profiles, as compared with the other donors. These signatures represent genes of potential importance for an immunoregulatory role of DC in an ongoing allergic response. © 2005 Blackwell Publishing Ltd.
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| 23. |
- Malmborg, Ann-Christin, et al.
(författare)
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Selection of binders from phage displayed antibody libraries using the BIAcore(TM) biosensor
- 1996
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 0022-1759. ; 198:1, s. 51-57
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Tidskriftsartikel (refereegranskat)abstract
- In this report we show that phage displayed antibodies can be selected based on dissociation rate constants, using a BIAcore(TM) biosensor. To demonstrate the principle, two Fab phage stocks displaying antibodies specific for hen egg lysozyme or phenyloxazolone were mixed in a ratio of 1:10 and injected over the biosensor chip containing immobilized lysozyme. Antigen-specific bound phages were eluted and analysed for specificity and phage titer. This procedure enriched for phages carrying specific antibodies. Selection of high affinity binders from phage libraries was then demonstrated with the BIAcore(TM) when phages were eluted and collected at different time points. Soluble antibody fragments were subsequently expressed and their kinetic parameters were determined. The time of elution was directly proportional to the affinity, due to decreased dissociation rate constants. This procedure offers a rapid and simple approach for selecting binders from phage libraries differing in antibody dissociation rate constants.
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| 24. |
- Nilsson, Nina, et al.
(författare)
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Altered gene expression associated with apoptosis in a pre-B-leukemic cell line following cross-linking of MHC class I
- 1997
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827. ; 231:1, s. 190-197
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Tidskriftsartikel (refereegranskat)abstract
- The major histocompatibility complex class I (MHC-I) has recently been shown not only to present antigens to the immune system but also to mediate transmembrane signaling, resulting in activation, inactivation, or apoptosis. Such signaling has been observed in both normal and malignant cells of the B and T cell lineage. Cross-linking of MHC-I on the pre-B- acute-lymphocytic cell line KM-3 induces an apoptotic process, which becomes evident after approximately 12 h. In order to better understand the mechanisms regulating this apoptotic process, we have investigated both gene expression and the effect of cross-linking on certain intracellular events. Differential display PCR was used to isolate two gene fragments whose level of expression was associated with the induction of apoptosis as they were downregulated in KM-3 cells following MHC-I cross-linking. These genes encode novel molecules whose function remains to be elucidated. It was further demonstrated that the apoptotic process was not accompanied by changes in [Ca2+](i), the level of activation of NF-κB, or changes in protein kinase C activity and that the initiation of apoptosis could be prevented by phorbol ester treatment. It is thus suggested that multiple, fine-tuned molecular events determine the outcome of cross-linking of MHC-I in this pre-B-lymphocytic cell line.
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| 25. |
- Ohlin, Mats, et al.
(författare)
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Characteristics of human antibody repertoires following active immune responses in vivo
- 1996
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Ingår i: Molecular Immunology. - : Elsevier BV. - 0161-5890. ; 33:7-8, s. 583-592
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Tidskriftsartikel (refereegranskat)abstract
- Possibilities to develop human monoclonal antibody specificities have recently been much facilitated by improvements of human hybridoma technology but even more so by the emerging phage-display technique. However, until recently very little has been known about the characteristics at the molecular level of the induced, T cell-dependent human antibody response, frequently targeted by these techniques. Rather, the major part of available sequence information has been related to tumor-derived or autoreactive antibodies. We have now investigated high affinity, monospecific, human antibody repertoires as developed by hybridoma technology. The VH region gene usage among such in vivo-induced repertoires is in only some respects similar to that found in the total B cell population. A limited number of heavy-chain variable segment loci account for the majority of all induced antibodies. A particular VH gene locus (4-34) frequently employed by peripheral B cells and associated with autoreactive antibodies was rarely used by the induced repertoire. Furthermore, in particular antigen systems, V region usage differs from the total available repertoire, and heavy-chain CDR3 is generally longer among antibodies induced against foreign protein antigens than in the average B cell population. Light-chain gene usage is often restricted to just a few dominant genes frequently found among B cells in general. In comparison, variable regions derived by phage-display technology in some antigen systems display even longer heavy-chain CDR3 than hybridoma-derived antibodies. This technique also appears to select a different set of germline genes preferentially (both with respect to VH and JH) as compared to hybridoma technology. In summary, the T cell-dependent antibody response against foreign antigens appears to differ from the average circulating B cell in several ways, and thus does not seem to represent a random selection of the available repertoire.
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| 26. |
- Ohlin, Mats, et al.
(författare)
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Cytomegalovirus glycoprotein B-specific antibody analysis using electrochemiluminescence detection-based techniques
- 1997
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Ingår i: Clinical and Diagnostic Laboratory Immunology. - 1071-412X. ; 4:1, s. 107-111
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Tidskriftsartikel (refereegranskat)abstract
- An electrochemiluminescence technique was used to develop versatile and sensitive assay strategies for determination of seroreactivities against biologically important cytomegalovirus neutralization epitopes expressed on glycoprotein B. Indirect binding assays showed wide linear assay ranges and revealed that serum samples diluted in parallel with a monoclonal antibody-based standard, simplifying quantitative analytical assessments.
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| 27. |
- Ohlin, M., et al.
(författare)
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Epstein-Barr virus-induced transformation of human B lymphocytes : the effect of l-leucyl-l-leucine methyl ester on inhibitory T cell populations
- 1992
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Ingår i: Immunology Letters. - 0165-2478. ; 34:3, s. 221-228
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Tidskriftsartikel (refereegranskat)abstract
- Epstein-Barr virus-mediated transformation of human B lymphocytes is inhibited by human T lymphocytes as well as by interferon-γ. Removal of the inhibitory cell populations is essential in order to achieve successful transformation in vitro. Cells with the capacity to inhibit outgrowth of lymphoblastoid cell lines can be removed by pretreatment of peripheral blood mononuclear cells with l-leucyl-l-leucine methyl ester. This treatment eliminates monocytes, NK-cells and a CD8+ T cell subpopulation. We now show that such treatment also has toxic effects on other human T cell populations. In addition, CD4+ and/or CD8+ lymphocytes are demonstrated to contain effector cell activities which inhibit outgrowth of EBV-transformed B cells. This inhibitory activity is abolished after treatment of peripheral blood mononuclear cells or purified CD4+ T cells with l-leucyl-l-leucine methyl ester. No evidence was found for a selective toxicity against any subset within the CD4+ or CD8+ T cell populations. However, the capacity of the treated cells, both peripheral blood mononuclear cells and purified CD4+ T lymphocytes, to produce mRNA encoding IFN-γ, a protein previously shown to downregulate outgrowth of EBV-transformed B cells, was selectively impaired. The results obtained suggest a role for CD4+ T cells to inhibit EBV-induced transformation of B cells.
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| 28. |
- Ohlin, M., et al.
(författare)
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Human antibody reactivity against the lower matrix protein (pp65) produced by cytomegalovirus
- 1995
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Ingår i: Clinical and Diagnostic Laboratory Immunology. - 1071-412X. ; 2:3, s. 325-329
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Tidskriftsartikel (refereegranskat)abstract
- The lower matrix protein (pp65) is a major product of many laboratory strains of cytomegalovirus (CMV). It is thus an integral part of many CMV serological assays based on native antigen. Recombinant fragments of pp65 have previously been investigated for their usefulness in more-defined assays. The latter antigens have, however, failed to develop a positive response with serum samples derived from a substantial number of infected individuals. Here we show that the human humoral immune response to CMV pp65 is highly diverse and recognizes at least seven distinct but in some cases partly overlapping epitopes. Most of these epitopes could not be mimicked by any of the investigated recombinant or synthetic antigens. Furthermore, when we investigated the ability of human CMV-seropositive serum samples to block the reactivity of pp65 specific antibodies recognizing five different epitopes within pp65, it was evident that several sera did not contain significant levels of antibodies against any of these or overlapping structures. It was thus concluded that the antibody response against CMV pp65 is weak in some CMV-infected individuals, making this antigen unsuitable for use alone in serological screening systems for CMV infection.
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| 29. |
- Ohlin, M., et al.
(författare)
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Human MoAbs produced from normal, HIV-1-negative donors and specific for glycoprotein gp120 of the HIV-1 envelope
- 1992
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Ingår i: Clinical and Experimental Immunology. - : Oxford University Press (OUP). - 0009-9104 .- 1365-2249. ; 89:2, s. 290-295
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Tidskriftsartikel (refereegranskat)abstract
- Human MoAbs ofIgM class were developed against three regions of the HIV-1 envelope. Uninfected donor lymphocytes were immunized in vitro with recombinant protein pB1. Four out of five antibodies were directed to different parts of the V3 region, which contains a major neutralizing site. Two out of these antibodies were directed to more than one amino acid sequence, indicating reactivity to discontinuous sites. Two of the human MoAbs inhibited viral spread between cells in tissue culture, interpreted as reactivities to conserved amino acid sequences exposed during viral maturation. No MoAb neutralized virus, which may be explained by the relatively low avidity of the antibodies. One MoAb was directed to a region containing amino acids participating in CD4 binding. This technique appears to allow formation of antibodies with fine specificities other than those obtained in infected hosts.
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| 30. |
- Ohlin, Mats, et al.
(författare)
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Insertions and deletions in hypervariable loops of antibody heavy chains contribute to molecular diversity
- 1998
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Ingår i: Molecular Immunology. - 0161-5890. ; 35:4, s. 233-238
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Tidskriftsartikel (refereegranskat)abstract
- Antibody diversity, a molecular feature which allows these proteins to specifically interact with a diverse set of targets, is created at the genetic level by a variety of means. These include germline gene segment recombination, junctional diversity and single basepair (bp) substitution. We here demonstrate that a human high affinity antibody specific for an exogenous protein antigen carry three amino acid residues immediately adjacent to the first hypervariable loop of the heavy chain. These additional residues are shown not to be encoded by the germline repertoire. We also describe the characteristics of insertions and deletions, not found in any known germline sequence, within the first and second hypervariable loops of other previously described antibody-encoding genes. These findings demonstrate that insertions or deletions of entire codons provide yet another approach by which the human antibody repertoire is diversified in vivo. Since these major genetic modifications occur within or immediately adjacent to loops contributing to the antigen-binding site, they are likely to affect the binding properties of the mutated antibodies.
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| 31. |
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| 32. |
- Ohlin, Mats, et al.
(författare)
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Low affinity, antibody binding of an Escherichia coli-derived component
- 1996
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Ingår i: FEMS Immunology and Medical Microbiology. - 0928-8244. ; 13:2, s. 161-168
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Tidskriftsartikel (refereegranskat)abstract
- This investigation describes the detection of a component in Escherichia coli capable of binding a large proportion of human antibody variable domains including otherwise highly monospecific antibodies induced by an in vivo antibody response. This interaction is of low affinity, but cross-linking of IgG molecules by, e.g. anti-immunoglobulin preparations, provides a sufficient degree of multivalency to promote a high avidity interaction. This binding which occurs both with K and h light chain-containing antibodies, appears to involve the variable region of human antibodies making it a superantigen-like activity. This is proposed based on the facts that: (i) different human antibodies of IgG1 isotype appear to bind to different extents suggesting that variable domain differences determine the binding activity; and (ii) addition of soluble antigen abrogates the interaction with the E. coli-derived molecule. Future studies of the nature and possible in vivo consequences of these interactions are warranted since any superantigen activity associated with this binding might affect the human immune response occurring as a consequence of E. coli infections.
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| 33. |
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| 34. |
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| 35. |
- Schoppel, K., et al.
(författare)
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Antibodies specific for the antigenic domain 1 of glycoprotein B (gpUL55) of human cytomegalovirus bind to different substructures
- 1996
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Ingår i: Virology. - : Elsevier BV. - 0042-6822. ; 216:1, s. 133-145
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Tidskriftsartikel (refereegranskat)abstract
- Glycoprotein B (gB, gpUL55) is a major antigen for the induction of neutralizing antibodies against human cytomegalovirus, making it an attractive antigen for active and passive immunoprophylaxis. The immunodominant region on gB is the antigenic domain 1 (AD-1), a complex structure which requires a minimal linear amino acid sequence of more than 75 amino acids (aa 552-635) for antibody binding. We have analyzed the fine specificity cf neutralizing and nonneutralizing AD-1-binding monoclonal antibodies. Point mutations were introduced into AD-1 and mutants were expressed as bacterial fusion proteins. The antigens were analyzed in immunoblots using a panel of 13 human and murine monoclonal antibodies. Complete loss of binding of all antibodies was observed with mutations at cysteine residues 573 and 610 as well as with a combinatorial exchange of prolines at position 577 and 613. The remaining mutations had different effects on antibody binding. Six individual recognition patterns were observed, indicating various antigenic substructures on AD-1. Changing the Fc portions of 3 murine monoclonal antibodies to human IgG1 showed that neutralization of AD-1-binding immunoglobulins is exerted by different mechanisms. Dependent on the recognized substructure within AD-1, avidity-dependent as well as Fc portion-mediated effects were observed.
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| 36. |
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