| 1. |
- Braide, M, et al.
(författare)
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Optimized density gradient separation of leukocyte fractions from whole blood by adjustment of osmolarity
- 1986
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 1872-7905 .- 0022-1759. ; 93:2, s. 183-191
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Tidskriftsartikel (refereegranskat)abstract
- Some of the compounds used for density gradient separation of blood cells have high osmolarities at the concentrations needed to create the required specific densities. Several mixed media use a combination of hyperosmolar shrinkage and red cell aggregation to improve cell separation. Due to the characteristics of Percoll density gradient medium the density and osmolarity of the gradient can be controlled separately. In the present study, Percoll gradients were used to determine the buoyant densities of different human blood cells at the osmolarities 300 mosM, 350 mosM and 400 mosM. Cell volumes were measured at the same osmolarities using a Coulter counter with channelyzer. As expected, the cell buoyant densities increased and the cell volumes decreased at the higher osmolarities used. There were, however, quantitative differences between the cells with respect to the effects of an increased osmolarity, making a 350 mosM density gradient the most effective in separating mononuclear leukocytes from polymorphonuclear leukocytes. A 400 mosM gradient offered the best possibilities to separate red blood cells from polymorphonuclear leukocytes. A one-step centrifugation procedure, based on these principles, is presented. This procedure makes possible the simultaneous purification of mononuclear leukocytes and polymorphonuclear leukocytes, suitable for functional assays.
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| 2. |
- Nilsson, Rune, et al.
(författare)
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Antigen-independent binding of rat immunoglobulins in a radioimmunoassay. Solutions to an unusual background problem
- 1984
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 1872-7905 .- 0022-1759. ; 66:1, s. 17-25
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Tidskriftsartikel (refereegranskat)abstract
- A high non-specific binding of immunoglobulins to plastic surfaces was noted with a number of rat sera, when tested in an indirect 125I-labeled protein-A assay for detection of cell-surface-bound rat immunoglobulins of various classes and IgG subclasses. This type of non-specific binding was found with all types of Ig. The degree of binding varied with the type of test plate used and fluctuated with time among sera drawn sequentially from the same donors. Coating test wells with fetal calf serum supplemented with BSA, gelatin or fibrinogen did not eliminate the reactions. The immunoglobulins bind directly to the polystyrene, and not to antigens present in fetal calf serum or autoantigens in rat serum. Two different approaches were used to eliminate the nonspecific reaction. When living cells were used as target antigens, exclusively cell-bound radioactivity was eluted with the non-ionic detergent Nonidet P40, which solubilizes the cell membrane without breaking the protein-A/rabbit IgG, rabbit IgG/rat Ig, or rat Ig/plastic interactions. When rat serum antibodies are tested on target antigens adsorbed on non-tissue culture grade plates, non-specific binding may be avoided by including 0.05% Tween 20 in the incubation mixture.
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| 3. |
- Nilsson, Rune, et al.
(författare)
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Quantitative analysis of rat Ig (sub) classes binding to cell surface antigens
- 1982
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 1872-7905 .- 0022-1759. ; 55:2, s. 179-191
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Tidskriftsartikel (refereegranskat)abstract
- An indirect 125I-labeled protein A assay for detection of cell surface-bound rat immunoglobulins is presented. The assay is quantitative and rapid and detects as little as 1 ng of cell surface-bound Ig. It discriminates between antibodies belonging to different IgG subclasses, IgM and IgA. We describe the production and specificity control of the reagents used and show that the test can be used for quantitative analysis. A large number of sera from untreated rats are tested to evaluate the frequency of falsely positive responses and variation due to age, sex and strain of rat. With this test it is relatively easy to quantitate the binding of classes and subclasses of rat immunoglobulins in a small volume (6 μl) of untreated serum.
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| 4. |
- Scharfstein, J, et al.
(författare)
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Antigenicity of cystatin-binding proteins from parasitic protozoan. Detection by a proteinase inhibitor based capture immunoassay (PINC-ELISA)
- 1995
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 1872-7905 .- 0022-1759. ; 182:1, s. 63-72
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Tidskriftsartikel (refereegranskat)abstract
- A novel immunoassay (PINC-ELISA) was designed using proteinase inhibitors of the cystatin superfamily (PINC) in the solid phase, to promote the selective capture of cysteine proteinases. The method was applied in the identification of papain-like antigens from parasitic protozoa. PINC of human origin, namely recombinant cystatin C (r-cystatin C) or low molecular weight kininogen were used in the assays to adsorb proteases contained in cell lysates from various trypanosomatids. The PINC-ELISA was at first optimized with the major cysteine proteinase from Trypanosoma cruzi (known as GP57/51 or cruzipain), an antigen whose serodiagnostic properties were previously established. Cruzipain is selectively adsorbed from crude extracts of T. cruzi onto PINC-coated wells; the finding that antibodies bind to epitopes located away from the sites of interaction with r-cystatin or low molecular weight kininogen has allowed for the screening of antibodies in chagasic sera, the methodology being advantageous in that it dispensed prior purification of the proteinase antigen. The PINC-ELISA was then carried out with lysates originating from Leishmania m. amazonensis (amastigotes) or Leishmania donovani (promastigotes). Complexes between solid-phase r-cystatin C and antigenic ligands in the lysates were again detected. The Leishmania molecules which bound to r-cystatin C, were respectively recognized by serum antibodies from mice chronically infected with L. amazonensis or from patients with visceral leishmaniasis. Direct evidence for the presence of cysteine proteinases in lysates from L. donovani was then obtained, using synthetic fluorogenic substrates. Due to the broad inhibition profile of r-cystatin C and the marked antigenicity of parasitic cysteine proteinases, such enzymes can be readily detected by PINC-ELISA, without requirement for prior knowledge of their substrate specificities.
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| 5. |
- Wadsworth, C, et al.
(författare)
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Spot immunoprecipitate assay in gel compared with immune reactions in solution and applied to antibody titration
- 1983
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 1872-7905 .- 0022-1759. ; 62:2, s. 217-229
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Tidskriftsartikel (refereegranskat)abstract
- Precipitation profiles of spot immunoprecipitate assay (SIA) reactions in gel are shown to have characteristics analogous to classic precipitin curves representing such immune reactions in solution. Thus SIA profiles of precipitated human albumin (HSA) and anti-HSA as checkerboard analysis permit: (1) determination of optimal proportions of antibody and antigen, (2) titration of antiserum as to antigen-binding capacity and (3) estimation of the concentration of specific antibody in an antiserum. The validity of the method was confirmed by similar results already reported for precipitin analysis of anti-HSA in solution. The stained protein assay method for determination of total protein used for the calculations had a reliable (r = 0.9979) working range for quantification between 0.1 and 0.83 microgram protein in 3 microliters samples (greater than or equal to 0.03 g/l). Study of SIA profiles in the extreme antibody excess region confirmed the validity of SIA quantification. The high sensitivity of SIA as compared with radial immunodiffusion is related to high ratios (8-9) of antibody to antigen; the reliable detection limit for SIA is 10 mg/l in a 3 microliters sample. SIA is easy to perform with simple laboratory equipment, allows measurement of any precipitable antigen with very small amounts of reactants, and gives results within an hour.
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| 6. |
- Andersson, C., et al.
(författare)
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Improved systems for hydrophobic tagging of recombinant immunogens for efficient iscom incorporation
- 2000
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Ingår i: JIM - Journal of Immunological Methods. - 0022-1759 .- 1872-7905. ; 238:02-jan, s. 181-193
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Tidskriftsartikel (refereegranskat)abstract
- We have previously reported a strategy for production in Escherichia coli of recombinant immunogens fused to a hydrophobic tag to improve their capacity to associate with an adjuvant formulation [Andersson et al., J. Immunol. Methods 222 (1999) 171]. Here, we describe a further development of the previous strategy and present significant improvements. In the novel system, the target immunogen is produced with an N-terminal affinity tag suitable for affinity purification, and a C-terminal hydrophobic tag, which should enable association through hydrophobic interactions of the immunogen with an adjuvant system, here being immunostimulating complexes (iscoms). Two different hydrophobic tags were evaluated: (i) a tag denoted M, derived from the membrane-spanning region of Staphylococcus aureus protein A (SpA), and (ii) a tag denoted MI consisting of the transmembrane region of hemagglutinin from influenza A virus. Furthermore, two alternative affinity tags were evaluated; the serum albumin-binding protein ABP, derived from streptococcal protein G, and the divalent IgG-binding ZZ-domains derived from SpA. A malaria peptide M5, derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, served as model immunogen in this study. Four different fusion proteins, ABP-MS-M, ABP-MS-MI, ZZ-MS-M and ZZ-MS-MI, were thus produced, affinity purified and evaluated in iscom-incorporation experiments. All of the fusion proteins were found in the iscom fractions in analytical ultracentrifugation, indicating iscom incorporation. This was further supported by electron microscopy analysis showing that iscoms were formed. In addition, these iscom preparations were demonstrated to induce MS-specific antibody responses upon immunisation of mice, confirming the successful incorporation into iscoms. The novel system for hydrophobic tagging of immunogens, with optional affinity and hydrophobic tags, gave expression levels that were increased ten to fifty-fold, as compared to the earlier reported system. We believe that the presented strategy would be a convenient way to achieve efficient adjuvant association for recombinant immunogens.
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| 7. |
- Ekerfelt, Christina, 1957-, et al.
(författare)
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Detection of spontaneous and antigen-induced human interleukin-4 responses in vitro : Comparison of ELISPOT, a novel ELISA and real-time RT-PCR
- 2002
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Ingår i: JIM - Journal of Immunological Methods. - 0022-1759 .- 1872-7905. ; 260:1-2, s. 55-67
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Tidskriftsartikel (refereegranskat)abstract
- Interleukin-4 (IL-4) is an important T-helper cell type 2 (Th2) cytokine in man, driving Th2 polarisation and exerting the most antagonistic effects to the Th1 cytokine interferon-? (IFN-?). Nevertheless, few data on spontaneous and antigen-specific secretion of IL-4 in man are available, mainly due to difficulties in the detection of IL-4. In this study, we compared three assays that can detect antigen-induced IL-4 responses, ELISPOT, ELISA after blocking the IL-4 receptor during cell culture, and real-time reverse transcription polymerase chain reaction (RT-PCR). Spontaneous, antigen- and allergen-induced responses were analysed in peripheral blood mononuclear cells from three groups with different secretion patterns for IL-4: atopic individuals, nonatopic individuals and pregnant women. ELISPOT displayed the highest sensitivity and was the only assay that could detect spontaneous secretion of IL-4 in all analysed samples. The IL-4 receptor blocking ELISA was considered best for the detection of in vitro antigen- and allergen-induced responses, since the results obtained from the ELISPOT and real-time RT-PCR displayed lower specificity, possibly because of seemingly aberrant IL-4 responses in the group of pregnant women. The real-time RT-PCR for detection of IL-4 mRNA proved to be sensitive, but expression of IL-4 mRNA was not correlated with the secretion of IL-4.
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| 8. |
- Jendeberg, L, et al.
(författare)
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Engineering of Fc(1) and Fc(3) from human immunoglobulin G to analyse subclass specificity for staphylococcal protein A.
- 1997
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Ingår i: JIM - Journal of Immunological Methods. - 0022-1759 .- 1872-7905. ; 201:1, s. 25-34
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Tidskriftsartikel (refereegranskat)abstract
- A system for production of recombinant Fc fragments of human IgG in Escherichia coli has been developed to allow for structural and functional studies of human Fc. The genes for the Fc fragments of human IgG subclasses 1 and 3, designated Fc(1) and Fc(3), were cloned from a human spleen cDNA library. The interactions to Staphylococcal protein A (SpA), a bacterial Fc receptor, that interacts with human IgG-Fc(1), but not with human IgG-Fc(3), were analyzed. To corroborate the involvement of amino acid residues in Fc, responsible for these differences in binding, two Fc variants were constructed; Fc(1(3)) and Fc(3(1)), each containing an isotypic dipeptide substitution. Production levels in E. coli of 1-10 mg/l of secreted Fc proteins, covalently linked as dimers, were routinely obtained. SpA-binding analyses of all four Fc variants using biosensor technology, showed that Fc(1) and Fc(3(1)) interact with SpA, while Fc(3) and Fc(1(3)) lack detectable SpA binding. The rendered SpA binding of the Fc variant Fc(3(1)), is concluded to result from the introduced dipeptide substitution (R435H, F436Y). The results demonstrate that the Fc expression system efficiently can be used in Fc engineering.
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| 9. |
- Lundqvist, Carina, et al.
(författare)
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Isolation of functionally active intraepithelial lymphocytes and enterocytes from human small and large intestine
- 1992
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Ingår i: JIM - Journal of Immunological Methods. - 0022-1759 .- 1872-7905. ; 152:2, s. 253-263
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Tidskriftsartikel (refereegranskat)abstract
- A mild purification method has been developed for the isolation of human intraepithelial lymphocytes (IEL) and enterocytes from the same individual. The isolation procedure includes mechanical disruption of the mucosal layer, treatment with reducing agent and sedimentation followed by Percoll gradient centrifugation. Finally, epithelial cells are removed from the IEL fraction using magnetic beads coated with the anti-epithelial antigen monoclonal antibody (mAb) BerEP4. Leucocytes are removed from the enterocyte fraction using magnetic beads coated with mAbs directed against common leucocyte antigen (CD45). Using this procedure IEL and enterocytes have been isolated from apparently normal jejunal, ileal and colonic tissue specimens. Recoveries of IEL were 7 x 10(5), 4 x 10(5) and 1 x 10(5)/cm2 mucosa from jejunum, ileum and colon respectively. 1-2 x 10(6) enterocytes/cm2 mucosa were recovered from small intestine while the corresponding value for colonic biopsies was approximately 2 x 10(5) enterocytes/cm2. The IEL fraction was pure as judged by the low percentages of B cells, macrophages and BerEP4 positive cells (less than 4%) present in the purified fraction. The enterocyte fraction contained less than 2% CD45+ cells. The two cell fractions were viable and expanded in vitro. Enterocytes expanded spontaneously while IEL required initial stimulation with mitogens. The isolation procedure described here will make it possible to study the function of human IEL, interactions between IEL and enterocytes and the role of both cell types in local immunity.
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| 10. |
- Ohlson, Sten, et al.
(författare)
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A Novel Approach to Monoclonal Antibody Separation Using High Performance Liquid Affinity Chromatography (HPLAC) with SelectiSpher-10 Protein G
- 1988
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 114, s. 175-180
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Tidskriftsartikel (refereegranskat)abstract
- Protein G, a bacterial cell wall protein extracted from strains of Streptococci, has been employed as a ligand in high performance liquid affinity chromatography (HPLAC) for separation of monoclonal antibodies. Examples are given of rapid high-resolution separations of rat and mouse monoclonal antibodies belonging to various subclasses. In comparison with protein A chromatography, we were able to show superior binding characteristics for SelectiSpher-10 protein G columns under conditions of 'low' ionic strengh (about 0.1 M) and neutral pH (pH ≈ 7). The monoclonal antibodies were isolated in high purity (> 90%) and with good recovery of specific activity (80–100%).We believe that the HPLAC technology based on SelectiSpher-10 protein G is of potential value in the analysis and purification of monoclonal antibodies from various species and subclasses.
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| 11. |
- Ohlson, Sten, et al.
(författare)
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Detecation of Circulating Immune Complexes by PEG Precipication Combinied with ELISA
- 1985
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 77:1, s. 87-93
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Tidskriftsartikel (refereegranskat)abstract
- An assay to detect circulating immune complexes is described and exemplified with serum from SLE and RA patients. The method is based on selective precipitation of immune complexes with PEG followed by a specific ELISA assay to detect the immunoglobulins present in the precipitate. The immunoglobulins of the precipitate were then monitored by their capacity to bind protein A and dDNA. With the PEG-protein A-ELISA procedure immune complexes were detected in 46% of the sera from SLE patients compared with 68% when using the PEG-DNA-ELISA.
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| 12. |
- Ohlson, Sten, et al.
(författare)
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Profiling of Circulating Immune Complexes by High Performance Liquid Affinity Chromatography (HPLAC) on a Protein A-Silica Matrix
- 1988
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 106:2, s. 225-229
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Tidskriftsartikel (refereegranskat)abstract
- Protein A, a cell wall protein found in bacterial strains of Staphylococcus aureus, has been extensively used in the analysis and purification of immunoglobulins and immune complexes. By binding protein A to microparticulate silica (high performance liquid affinity chromatography, HPLAC), a rapid and efficient chromatographic system was obtained for the separation and analysis of circulating immune complexes. The method was applied to the separation of artificial immune complexes as well as to plasma samples from patients with immune complex associated diseases such as SLE and RA. It was possible to distinguish certain subpopulations of circulating immune complexes by performing pH gradients on the protein A silica HPLAC column.
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| 13. |
- Ronnmark, J., et al.
(författare)
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Construction and characterization of affibody-Fc chimeras produced in Escherichia coli
- 2002
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Ingår i: JIM - Journal of Immunological Methods. - 0022-1759 .- 1872-7905. ; 261:02-jan, s. 199-211
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Tidskriftsartikel (refereegranskat)abstract
- Affibody-Fc chimeras were constructed by genetic fusion between different affibody affinity proteins with prescribed specificities and an Fc fragment derived from human I-G. Using affibody ligands previously selected for binding to respiratory syncytial virus (RSV) surface protein G and Thermus aquaticus (Taq) DNA polymerase, respectively, affibody-Fc fusion proteins showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges were produced in Escherichia coli and affinity purified on protein A Sepharose from bacterial periplasms at yields ranging between 1 and 6 mg/l culture, Further characterization of the chimeras using biosensor technology showed that the affibody moieties have retained high selectivities for their respective targets after fusion to the Fc fragment. Avidity effects in the target binding were observed for the affibody-Fc chimeras compared to monovalent affibody fusion proteins, indicating that both affibody moieties in the chimeras were accessible and contributed in the binding. Fusion of a head-to-tail dimeric affibody moiety to the Fc fragment resulted in tetravalent affibody constructs which showed even more pronounced avidity effects. In addition, the Fc moiety of the chimeras was demonstrated to be specifically recognized by anti-human IgG antibody enzyme conjugates. One application for this class of artificial antibodies was demonstrated in a western blotting experiment in which one of the anti-RSV surface protein G affibody-Fc chimeras was demonstrated to be useful for specific detection of the target protein in a complex background consisting of a total E. coli lysate. The results show that through the replacement of the Fab portion of an antibody for an alternative binding domain based on a less complicated structure, chimeric proteins compatible with bacterial production routes containing both antigen recognition domains and Fc domains can be constructed. Such artificial antibodies should be interesting alternatives to, for example, whole antibodies or scFv-Fc fusions as detection devices and in diagnostic or therapeutic applications.
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| 14. |
- Svanholm, Cecilia, et al.
(författare)
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Enhancement of antibody responses by DNA immunization using expression vectors mediating efficient antigen secretion
- 1999
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Ingår i: JIM - Journal of Immunological Methods. - 0022-1759 .- 1872-7905. ; 228:1-2, s. 121-130
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Tidskriftsartikel (refereegranskat)abstract
- The immune responses elicited in mice, after intradermal (i.d.) immunization with plasmids encoding secreted or intracellular forms of HIV-1 nef, HIV-1 tat or C. pneumoniae omp2 proteins, respectively, were compared. To mediate secretion of these proteins the genes were fused to a heterologous signal sequence from murine heavy chain IgG. The nef- and omp2-specific antibody responses were dramatically increased when mice were inoculated with the plasmid encoding the secreted form of these proteins. In contrast, HIV-1 tat comprising an internal strong nuclear targeting sequence could not be induced to secretion and subsequently no enhanced antibody response was observed. Slight improvement of the HIV-1 nef antibody response was achieved after co-inoculation with a granulocyte-macrophage colony-stimulating factor (GM-CSF) expression vector. Further, nef-specific T-cell responses were induced after nef DNA injections, and were of Th1-like phenotype regardless of whether the nef protein was secreted or not. The system described in this study, using a plasmid vector with a strong heterologous signal sequence that mediate efficient antigen secretion in vivo, may have wide applicability for the induction of high antibody levels to normally non-secreted antigens.
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| 15. |
- Ullén, Anders, et al.
(författare)
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In vivo and in vitro interactions between idiotypic and antiidiotypic monoclonal antibodies against placental alkaline phosphatase
- 1995
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Ingår i: JIM - Journal of Immunological Methods. - 0022-1759 .- 1872-7905. ; 183:1, s. 155-165
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Tidskriftsartikel (refereegranskat)abstract
- A monoclonal antiidiotypic antibody alpha H7, was generated against a monoclonal antibody H7 with specificity towards placental alkaline phosphatase. The in vitro and in vivo effects of alpha H7 were investigated. The antiidiotypic antibody was found to generate stable complexes with the radiolabeled idiotypic antibody, visualized both in vivo and in vitro, as revealed by PAGE and autoradiography. Using biosensor technology (BIAcore, Pharmacia) the interactions were followed in real time and the association rate, dissociation rate, and affinity constants between the reactants were determined (KA H7/PLAP 6.7 x 10(9) M-1, KA H7/alpha H7 3.2 x 10(9) M-1). By in vivo injection of the antiidiotype, a rapid dose dependent clearance of circulating radiolabeled idiotypes was demonstrated and a decrease in total body radioactivity was recorded with a concomitant dramatic increase in non-protein-bound 125I excreted in the urine. It is concluded that idiotypic-antiidiotypic interactions offer advantages in the regulation of antibody levels in vivo.
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| 16. |
- Whiss, Per A., et al.
(författare)
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Modulation of P-selectin expression on isolated human platelets y an NO donor assessed by a novel ELISA application
- 1997
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Ingår i: JIM - Journal of Immunological Methods. - 0022-1759 .- 1872-7905. ; 200:1-2, s. 135-143
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Tidskriftsartikel (refereegranskat)abstract
- Adhesion molecules such as P-selectin are potential markers for evaluating platelet activation and studying the role of cell-cell interactions in numerous biological processes related to hemostasis and inflammation. The expression of P-selectin and related molecules has previously been quantified with different techniques. As an alternative to the most common method, flow cytometry, we have developed a useful ELISA method to simultaneously analyse 96 samples for platelet expression of P-selectin. Samples may be stored for at least 7 days at 4°C prior to analysis. The method is simple, reproducible, flexible and requires only standard equipment. Washed platelets (WP) from healthy male volunteers, at a concentration of 1 × 107/microtiter plate well, were stimulated with various known platelet activators and fixed with 0.1% formaldehyde for 10 min. The fixed WP were centrifuged to form a confluent layer in the wells and then incubated with optimal dilutions of primary antibodies (1/2000) directed against P-selectin, CD41, CD9 and secondary antibodies conjugated with alkaline phosphatase. Our results show that P-selectin expression on WP increases significantly upon stimulation with thrombin (0.1–1.0 U/ml), ADP (10 μM) and epinephrine (100 μM). The induction of P-selectin expression by thrombin is fast and has different kinetics depending on the concentration of the agonist. Prior incubation with the nitric oxide donor SNAP (10 μM) inhibits the up-regulation of P-selectin induced by sub-maximal concentrations of thrombin (p < 0.05). This ELISA is suitable for studying the expression and regulation of P-selectin and other surface molecules on human platelets in various pathological states.
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| 17. |
- Zhao, Linshu, et al.
(författare)
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Purification and characterization of a 95 kDa protein : from normal human granulocytes
- 2002
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Ingår i: JIM - Journal of Immunological Methods. - 0022-1759 .- 1872-7905. ; 270:1, s. 27-35
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Tidskriftsartikel (refereegranskat)abstract
- A 95-kDa protein was purified to homogeneity from granule extracts of normal human granulocytes. The column procedure consisted of Sephadex G-75, Mono-S cation exchange and Superdex HR 75 chromatography. The purified protein showed only one broad band at a molecular weight of 95 kDa when analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It reacted with polyclonal antibodies against carcinoembryonic antigen (CEA) and a specific monoclonal antibody against CD66b, but did not react with monoclonal antibodies against CD66acde and CD66c when analyzed by immunoblotting. The molecular weight of the protein shifted from 95 to 40 kDa on SDS-PAGE after deglycosylation. Tryptic peptide analysis by MALDI-Tof identified four peptides with spectra of m/z matching the expected tryptic peptides from a CGM6 gene product. Furthermore, the nanoelectrospray mass spectrometry (MS/MS) analysis of the two selected tryptic peptides of the protein revealed two amino acid sequences corresponding to residues 79-98 and 199-207 of the CGM6 gene product. Based on this, and also on the immunochemical data, it is concluded that the purified 95 kDa is identical to carcinoembryonic antigen-related cell adhesion molecule 8 (CEACAM8) (nonspecific cross-reacting antigen (NCA)-95, CD67 and CD66b) and is a product of the CGM6 (W272) gene. We present, for the first time, a method for the purification of CEACAM8 from normal human granulocytes, which should be useful for further studies on its structure and functions. We also confirmed at the protein level that CEACAM8 is a product of the CGM6 (NCA-W272) gene.
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| 18. |
- Afonso, Georgia, et al.
(författare)
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Critical parameters in blood processing for T-cell assays: Validation on ELISpot and tetramer platforms
- 2010
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 1872-7905 .- 0022-1759. ; 359:1-2, s. 28-36
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Tidskriftsartikel (refereegranskat)abstract
- Assays detecting antigen (Ag)-specific T-cell responses in immune-mediated processes are increasingly employed to understand disease pathogenesis and "immune staging". The quantity and quality of starting peripheral blood mononuclear cell (PBMC) preparations are important factors in the performance of such assays. We therefore compared final PBMC yield and function by modifying parameters at the blood drawing, storage and processing steps. While drawing blood in vacuum-driven tubes or syringes and separating PBMCs on density gradients using standard or membrane (Leucosep (R)) tubes made no difference, storing tubes for 18 h without any agitation led to PBMC preparations contaminated with granulocytes and decreased interferon (IFN)-gamma enzyme-linked immunospot (ELISpot) responses. Even agitated blood showed a trend towards reduced ELISpot responses and increased human leukocyte Ag (HLA) multimer readouts when stored for 18 h compared to 3 h. These changes were reduced by diluting blood prior to storage. Washing PBMCs with media containing 10% human serum increased PBMC yields by 40.5%, without affecting ELISpot responses and multimer counts. However, washes with >10% human serum decreased multimer counts, with no additional improvement in PBMC yields. These findings may be relevant for optimizing and harmonizing PBMC processing procedures for T-cell assays. (C) 2010 Elsevier B.V. All rights reserved.
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| 19. |
- Andersson, M., et al.
(författare)
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Inclusion of a non-immunoglobulin binding protein in two-site ELISA for quantification of human serum proteins without interference by heterophilic serum antibodies
- 2003
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 283:02-jan, s. 225-234
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Tidskriftsartikel (refereegranskat)abstract
- Measurement of human serum molecules with two-site ELISA can be biased by the presence of human heterophilic anti-animal immunoglobulin antibodies (HAIA) that cause false-positive signals by cross-linking the monoclonal (mAb) and/or polyclonal antibodies (pAb) used for the pre- (capture) and post-analyte steps (detection). To evaluate a novel ELISA format designed to avoid interference by HAIA, a target-specific non-immunoglobulin (Ig) affinity protein (affibody) was used to replace one of the antibodies. First, a human IgA-binding affibody (Z(IgA)) selected by phage display technology from a combinatorial library of a single Staphylococcus aureus protein A domain was used. The detection range of IgA standard using an ELISA based on Z(IgA) for capture and goat pAb against IgA (pAb(IgA)) for detection was comparable with that of using pAb(IgA) for both capture and detection. Secondly, another affibody (Z(Apo)) was combined with mAb and used to detect recombinant human apolipoprotein A-1. The affibody/antibody ELISAs were also used to quantify human serum levels of IgA and apolipoprotein A1. To verify that human serum did not cause false-positive signals in the affibody/antibody ELISA format, the ability of human serum to cross-link affibodies, mAb (mouse or rat) and/or pAb (goat) displaying non-matched specificities was assessed; affibodies and antibodies were not cross-linked whereas all combinations of mAb and/or pAb were cross-linked. The combination of affibodies and antibodies for analysis of human serum molecules represents a novel two-site ELISA format which precludes false-positive signals caused by HAIA.
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| 20. |
- Areström, Irene, et al.
(författare)
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Measurement of human latent transforming growth factor beta 1 using a latency associated protein reactive elisa
- 2012
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 379:1-2, s. 23-29
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Tidskriftsartikel (refereegranskat)abstract
- Human Transforming Growth Factor (TGF)-beta 1, one of three TGF-beta isoforms, is a pleotropic cytokine critical for many physiological and immunological processes. TGF-beta 1 is secreted in a latent form, linked to Latency Associated Protein (LAP). Analysis of Latent TGF-beta 1 by TGF-ELISA requires dissociation of TGF-beta 1 from LAP, e.g. by acidification of samples. The ELISA then measures total TGF-beta 1, equivalent to dissociated Latent TGF-beta 1 plus any free TGF-beta 1 present prior to acidification. Evolutionary conservation of TGF-beta 1 across mammals also renders TGF-beta 1 ELISAs reactive with TGF-beta 1 in bovine serum often used in human cell cultures. To enable a direct analysis of Latent TGF-beta 1, monoclonal antibodies were made against LAP from human latent TGF-beta 1 and used to develop a LAP ELISA detecting Latent TGF-beta 1. The ELISA did not react with LAP from human Latent TGF-beta 2 or 3, respectively, nor with Latent TGF-beta in bovine serum. EDTA-containing plasma from healthy subjects (n = 20) was analyzed by conventional TGF-beta 1 ELISA and LAP ELISA. By TGF-beta 1 ELISA, total TGF-beta 1 were detected in all samples (median 133 pM, range 34-348 pM); low levels of free TGF-beta 1 found in 8/20 non-addified samples showed that >98.5% of the total TGF-beta 1 derived from Latent TGF-beta 1. Latent TGF-beta 1 found in non-acidified samples by LAP ELISA (median 154 pM, range 48-403 pM) was comparable in molar levels to, and correlated with, total TGF-beta 1 (r(s) 0.96, p<0.0001). A similar agreement between the total TGF-beta 1 and the LAP ELISA was found with citrate- and heparin-containing plasma. The LAP ELISA facilitates analysis of Latent TGF-beta 1 without sample acidification and is not compromised by the presence of bovine serum in human cell supernatants.
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| 21. |
- Backteman, Karin, 1960-, et al.
(författare)
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Biological and methodological variation of lymphocyte subsets in blood of human adults
- 2007
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 322:1-2, s. 20-27
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Tidskriftsartikel (refereegranskat)abstract
- Although lymphocyte populations are often monitored over time, information about the biological variation over time is limited. Three-colour-flow cytometry was used to investigate the biological and methodological variation of lymphocyte populations in blood. Fifteen healthy individuals (11 females and 4 males) were longitudinally monitored for 2-8 years. Blood samples were drawn monthly when possible. In total, 493 observations were included. Absolute counts and proportions were determined for T-cells (CD3+), T-helper cells (CD3+ CD4+), cytolytic T-cells (CD3+ CD8+), B-cells (CD3- CD19+) and NK-cells (CD3- CD16+/56+). As to variation over the year, ANOVA testing showed only a minor monthly variation for absolute counts of the CD8+ population (p < 0.05) for October compared with June and July, whereas no significant differences were found for the other populations or in the proportions of lymphocyte subsets. Although lower than the longitudinal variation, the methodological variation, expressed as coefficient of variation (CV %), was in a similar range as the variation over time, indicating that the normal biological variation should not be overestimated, while the methodological inter-assay should be taken into consideration in longitudinal studies or monitoring of patients. © 2007 Elsevier B.V. All rights reserved.
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| 22. |
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| 23. |
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| 24. |
- Carlsson, Fredrika, et al.
(författare)
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A dimerized single-chain variable fragment system for the assessment of neutralizing activity of phage display-selected antibody fragments specific for cytomegalovirus.
- 2012
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 1872-7905 .- 0022-1759. ; 376:Online 01 Dec 2011, s. 69-78
-
Tidskriftsartikel (refereegranskat)abstract
- Abstract in UndeterminedCytomegalovirus (CMV) causes severe sequelae in congenitally infected newborns and may cause life-threatening disease in immuno-deficient patients. Recent findings demonstrate the possibility to alleviate the disease by infusing intravenous immunoglobulin G (IgG) preparations, indicating that antibodies are an effective therapeutic option. Modern molecular methodologies, like phage display, allow for the development of specific antibodies targeting virtually any antigen, including those of CMV. However, such methodologies do not in general result in products that by themselves mediate biological activity. To facilitate a semi-high-throughput approach for functional screening in future efforts to develop efficacious antibodies against CMV, we have integrated two different approaches to circumvent potential bottlenecks in such efforts. Firstly, we explored an approach that permits easy transfer of antibody fragment encoding genes from commonly used phage display vectors into vectors for the production of divalent immunoglobulins. Secondly, we demonstrate that such proteins can be applied in a novel reporter-based neutralization assay to establish a proof-of-concept workflow for the generation of neutralizing antibodies against CMV. We validated our approach by showing that divalent antibodies raised against the antigenic domain (AD)-2 region of gB effectively neutralized three different CMV strains (AD169, Towne and TB40/E), whereas two antibodies against the AD-1 region of gB displayed minor neutralizing capabilities. In conclusion, the methods investigated in this proof-of-concept study enables for a semi-high-throughput workflow in the screening and investigation of biological active antibodies.
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| 25. |
- Clinchy, Birgitta, et al.
(författare)
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Differences in adsorption of serum proteins and production of IL-1ra by human monocytes incubated in different tissue culture microtiter plates
- 2003
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 282:1-2, s. 53-61
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Tidskriftsartikel (refereegranskat)abstract
- In vitro cell culture models can be of great value in order to further analyze the regulatory mechanisms underlying the inappropriate function of the immune system in diseases such as autoimmunity and cancer. Cell culture conditions have to be well controlled in a way that they mirror the in vivo situation. The objective of this study was to compare tissue culture microtiter plates from different manufacturers with respect to their ability to support monokine production by human monocytes cultured in human serum. Tissue culture ware, made of polystyrene, undergoes treatment by the manufacturers to make the surface more suitable for culture of adherent cell populations. It is possible that quality differences in this treatment can lead to variations in protein binding properties and thereby influence the adherence and functional properties of monocytes. We measured spontaneous interleukin-1 receptor antagonist (IL-1ra) production by peripheral blood monocytes, cultured in human serum, in five different microtiter plates made for adherent cell culture. Culture in plates from two of the five manufacturers resulted in significantly lower amounts of secreted IL-1ra. IL-1ra release by human monocytes can be induced by adherent IgG cross-linking membrane receptors for the Fc part of IgG (Fc?R). We found that reduced IL-1ra production coincided with a reduced capacity for binding of serum IgG in one case. Furthermore, this brand of microtiter plate also displayed the lowest level of adsorption of human albumin. We conclude that the protein adsorption properties of the plastic tissue culture ware have to be taken into consideration when assessing monokine production by human monocytes in vitro.
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| 26. |
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| 27. |
- Davidsson, Lisa, et al.
(författare)
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A simple skin blister technique for the study of in vivo transmigration of human leukocytes.
- 2013
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Ingår i: Journal of immunological methods. - : Elsevier BV. - 1872-7905 .- 0022-1759. ; 393:1-2, s. 8-17
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Tidskriftsartikel (refereegranskat)abstract
- The study of human leukocytes is almost exclusively conducted using cells isolated from peripheral blood. This is especially true for neutrophils, despite the fact that these cells are of main (pathological) importance in extravascular tissues upon e.g., infection and/or tissue damage. The journey from circulation to tissue is typically associated with a number of cellular changes, making the cells primed, or hyper-responsive, and in many aspects distinct from the cells present in circulation. Models to obtain in vivo transmigrated leukocytes from human tissue are available, but not widely used. We describe here an easy-to-use model for the study of local inflammation, stemming from limited tissue damage, which can be used to isolate viable and functional leukocytes. The model is based on the generation of aseptic skin blisters, formed by the application of negative pressure, and allows for investigations of the cellular infiltrate as well as of soluble mediators present in the exudate. We believe that this method, combined with modern analysis equipment suitable for small volumes and cell numbers, could be of great use for increasing our understanding of the nature and function of leukocytes that have left circulation and transmigrated to inflamed tissues.
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| 28. |
- Elshafie, Amir, 1970-, et al.
(författare)
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General false positive ELISA reactions in visceral leishmaniasis. Implications for the use of enzyme immunoassay analyses in tropical Africa.
- 2016
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 431, s. 66-71
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Tidskriftsartikel (refereegranskat)abstract
- Leishmaniasis is a neglected disease in tropical countries. Clinical and laboratory features may mimic auto immune diseases and this can complicate the Leishmania diagnosis. Due to our previous investigation for false anti-CCP2 reactivity in Leishmania-infected subjects and our interest in immunity against the joint-specific collagen type II (CII) in rheumatoid arthritis (RA) we investigated the same cohort for anti-CII antibodies. We found elevated anti-CII reactivity in Leishmania-infected patients as compared to controls. When anti-CII OD values were compared with BSA-blocked control plates we found higher reactivity against BSA than in CII-coated plates in many Leishmania-infected patients. The percentage of such false positive anti-CII reactions increased with inflammatory activity, and was found in almost all Leishmania patients with highly active inflammatory disease, but was as low in Sudanese healthy controls as well as among Swedish RA patients. The correlation coefficients between false positive anti-CII and anti-CCP2 measured with a commercial ELISA were highest for patients with the most inflammatory disease but non-significant for Sudanese controls and Swedish RA patients, arguing that our findings may have general implications for ELISA measurements in leishmaniasis. ELISA investigations in areas endemic for leishmaniasis might benefit from individual-specific control wells for each serum sample. This approach might also be applicable to other geographical areas or patient groups with high incidence of inflammatory and infectious diseases.
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| 29. |
- Engström, Henrik, et al.
(författare)
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Towards a FRET-based immunosensor for continuous carbohydrate monitoring
- 2008
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 333:1-2, s. 107-114
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Tidskriftsartikel (refereegranskat)abstract
- In this report we have evaluated the potential of using fluorescence/Förster resonance energy transfer (FRET) in a competitive immunosensor for continuous monitoring of the carbohydrate hapten maltose. The cyanine dyes Cy5 and Cy5.5 were used as a donor–acceptor pair by conjugation to maltose-labeled bovine serum albumin (BSA) and the monoclonal antibody IgG 39.5, giving Cy5–BSA–maltotriitol (3.1/1/18) and Cy5.5–mAb39.5 (2.2/1), respectively. This antibody with weak affinity towards maltose showed full reversibility to both the free maltose and the maltose-labeled conjugate. It allowed us to measure continuously the maltose content by monitoring the FRET signal change over time due to displacement of Cy5–BSA–maltotriitol from Cy5.5–mAb39.5 inside a semipermeable capsule. A near 22% total increase was seen in the fluorescence intensity ratio I670/I700 in the presence of maltose, with a calculated EC50 = 1.87 ± 0.13 mM (R2 = 0.9984) from the sigmoidal dose–response curve at 25 °C. Specificity of the immunosensor was shown with the structural analog to maltose, cellobiose, and it generated no detectable response. A minor drift in the sensor baseline was seen with 0.4% per 24 h, which was in the same magnitude as the signal-to-noise ratio, during the 4 weeks of measurements. The immunosensor was applied to crude samples of oat drinks for direct quantification of the maltose content. Overall, this work demonstrates the potential to use an immunosensor based on weakly binding antibodies and FRET technology for remote and non-invasive carbohydrate monitoring.
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| 30. |
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| 31. |
- Feldhahn, Magdalena, et al.
(författare)
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miHA-Match : Computational detection of tissue-specific minor histocompatibility antigens
- 2012
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier. - 0022-1759 .- 1872-7905. ; 386:1-2, s. 94-100
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Tidskriftsartikel (refereegranskat)abstract
- Allogenic stem cell transplantation has shown considerable success in a number of hematological malignancies, in particular in leukemia. The beneficial effect is mediated by donor T cells recognizing patient-specific HLA-binding peptides. These peptides are called minor histocompatibility antigens (miHAs) and are typically caused by single nucleotide polymorphisms. Tissue-specific miHAs have successfully been used in anti-tumor therapy without causing unspecific graft-versus-host reactions. However, only a small number of miHAs have been identified to date, limiting the clinical use.Here we present an immunoinformatics pipeline for the identification of miHAs. The pipeline can be applied to large-scale miHA screening, for example, in the development of diagnostic tests. Another interesting application is the design of personalized miHA-based cancer therapies based on patient-donor pair-specific miHAs detected by this pipeline. The suggested method covers various aspects of genetic variant detection, effects of alternative transcripts, and HLA-peptide binding. A comparison of our computational pipeline and experimentally derived datasets shows excellent agreement and coverage of the computationally predicted miHAs.
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| 32. |
- Follin, Per
(författare)
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Skin-chamber technique for study of in vivo exudated human neutrophils
- 1999
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Ingår i: JIM - Journal of Immunological Methods. - 0022-1759 .- 1872-7905. ; 232:1-2, s. 55-65
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Tidskriftsartikel (refereegranskat)abstract
- The development of new techniques for isolation of neutrophils extravasated in vivo have been essential for studying the dynamics of the inflammatory response in humans. Methods for generating inflammatory skin reactions were first presented in the mid 1950s, and later a skin blistering technique based on suction was introduced. With this procedure, small areas of denuded dermis, called "skin windows", are created and covered with special chambers containing a medium that attracts exudated neutrophils. By comparing the neutrophils collected in such chambers with those isolated from peripheral blood, it is possible to investigate the functional modifications that neutrophils undergo when attracted to an inflammatory process. The skin-blister chamber technique represents an aseptic, non-traumatic and reproducible model of inflammation that can be used to study in vivo activated human neutrophils. The background, methodological aspects and options of this technique are described, together with the functional characteristics of exudated neutrophils. (C) 1999 Elsevier Science B.V. All rights reserved.
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| 33. |
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| 34. |
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| 35. |
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| 36. |
- Holthaus, Lisa, et al.
(författare)
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CD4(+) T cell activation, function, and metabolism are inhibited by low concentrations of DMSO
- 2018
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 463, s. 54-60
-
Tidskriftsartikel (refereegranskat)abstract
- Dimethyl sulfoxide (DMSO) is a polar organic solvent used in a wide range of biological applications. DMSO is routinely used as a cryoprotectant for long-term cell freezing as well as to dissolve peptides and drugs for immune cell functional assays. Here, human CD4(+) T cell activation, cytokine production, proliferation, and metabolism were investigated after stimulation in the presence of 0.01% to 1%, DMSO, representing concentrations commonly used in vitro. Surface expression of the activation markers CD69, CD25 and CD154 after polyclonal activation of CD4(+) T cells was inhibited by 0.25% or higher concentrations of DMSO. The frequencies of IL-21(+), IL-4(+), and IL-22(+) CD4(+) T cells, following polyclonal activation were variably inhibited by DMSO at concentrations ranging from 0.25% to 1%, whereas IFN gamma(+) cells were unaffected. CD4(+) T cell proliferation after anti-CD3 or antigen stimulation was inhibited by 0.5% DMSO and abolished by 1% DMSO. After polyclonal stimulation, glucose uptake was inhibited in the presence of 1% DMSO, but only minor effects on CD4+ T cell respiration were observed. Consistent with the immune effects, the gene expression of early signaling and activation pathways were inhibited in CD4+ T cells in the presence of 1% DMSO. Our study revealed that DMSO at concentrations generally used for in vitro studies of T cells impacts multiple features of T cell function. Therefore, we urge care when adding DMSO-containing preparations to T cell cultures.
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| 37. |
- Huang, Jenny, et al.
(författare)
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ELISpot and ELISA analyses of human IL-21-secreting cells : Impact of blocking IL-21 interaction with cellular receptors
- 2015
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 417, s. 60-66
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Tidskriftsartikel (refereegranskat)abstract
- Interleukin (IL)-21 is crucial for the regulation of lymphocytes and is implicated in autoimmune and other diseases. The relevance of being able to measure human IL-21 prompted us to develop ELISA and ELISpot assays for analysis of IL-21 levels and IL-21-producing cells, respectively. Monoclonal antibodies (mAbs) to IL-21 were made and ELISA and ELISpot assays were developed. The selected detection mAb also neutralized IL-21-mediated activation of human cells. Peripheral blood mononuclear cells (PBMCs) from healthy donors (n = 24) were stimulated polyclonally (phytohemagglutinin; PHA) or with antigen (Candida albicans extract and tetanus toxoid). Using ELISpot, high numbers of IL-21-producing cells were detected after PHA activation; lower but positive responses to antigen were seen in approximately 50% of the donors. In contrast, the ELISA detected IL-21 in supernatants from PHA-activated cells but not from antigen-stimulated cells. When analyzing IL-17A in parallel, PHA and antigens induced detectable responses in ELISpot as well as in ELISA. Hypothesizing that the lack of detectable IL-21 levels after antigenic stimulation was due to a combination of low frequencies of IL-21-secreting cells and consumption of IL-21 by cellular receptors during cell culture, PBMCs (n = 18) were stimulated in the presence of the neutralizing detection mAb. When preventing IL-21 from interacting with its receptor, increased IL-21 levels were found by ELISA after PHA activation and IL-21 could also be measured after antigen stimulation. ELISpot results were unaffected by the addition of the neutralizing mAb. In conclusion, IL-21 secreted by low frequencies of antigen-specific ex vivo-stimulated PBMC can be difficult to detect by ELISA but prevention of IL-21 interaction with its receptor leads to detectable IL-21 levels. In ELISpot, where the cytokine is captured by mAbs on a solid phase immediately upon secretion, blocking the receptor interaction does not affect the detection of IL-21-secreting cells.
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| 38. |
- Höglind, Ankie, et al.
(författare)
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Systematic evaluation of monoclonal antibodies and immunoassays for the detection of Interferon-gamma and Interleukin-2 in old and new world non-human primates
- 2017
-
Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 441, s. 39-48
-
Tidskriftsartikel (refereegranskat)abstract
- Non-human primates (NHP) provide important animal models for studies on immune responses to infections and vaccines. When assessing cellular immunity in NHP, cytokines are almost exclusively analyzed utilizing cross-reactive anti-human antibodies. The functionality of antibodies has to be empirically established for each assay/application as well as NHP species.A rational approach was employed to identify monoclonal antibodies (mAb) cross-reactive with many NHP species. Panels of new and established mAbs against human Interferon (IFN)-gamma and Interleukin (IL)-2 were assessed for reactivity with eukaryotically expressed recombinant IFN-gamma and IL-2, respectively, from Old (rhesus, cynomolgus and pigtail macaques, African green monkey, sooty mangabey and baboon) and New World NHP (Ma's night monkey, squirrel monkey and common marmoset).Pan-reactive mAbs, recognizing cytokines from all NHP species, were further analyzed in capture assays and flow cytometry with NHP peripheral blood mononuclear cells (PBMC). Pan-reactive mAb pairs for IFN-gamma well as IL-2 were identified and used in ELISA to measure IFN-gamma and IL-2, respectively, in Old and New World NHP PBMC supernatants. The same mAb pairs displayed high functionality in ELISpot and FluoroSpot for the measurement of antigen-specific IFN-gamma and IL-2 responses using cynomolgus PBMC Functionality of pan-reactive mAbs in flow cytometry was also verified with cynomolgus PBMC.The development of well-defined immunoassays functional with a panel of NHP species facilitates improved analyses of cellular immunity and enables inclusion in multiplex cytokine assays intended for a variety of NHP.
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| 39. |
- Jafari, Rozbeh, 1977-, et al.
(författare)
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Construction of divalent anti-keratin 8 single-chain antibodies (sc(FV)2), expression in Pichia Pastoris and their reactivity with multicellular tumor spheroids
- 2011
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 364:1-2, s. 65-76
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Tidskriftsartikel (refereegranskat)abstract
- Single-chain variable fragments (scFvs) are small monovalent recombinant antibody fragments that retain the specificity of their parent immunoglobulins. ScFvs are excellent building blocks for new and improved immunodiagnostic and therapeutic proteins. However, the monovalency and the rapid renal elimination of scFvs result in poor tumor accumulation and retention. Engineering divalent antibody fragments is an excellent way to address these shortcomings. In this study, covalent divalent single-chain variable fragments (sc(Fv)2s), were constructed from the monovalent anti-keratin 8 scFvs, TS1-218 and its mutant, HE1-Q. The scFvs and sc(Fv)2s were expressed in the methylotrophic yeast Pichia pastoris, utilizing the alpha-factor secretion signal (α-factor) for extracellular secretion. The immunoreactivity and specificity of the antibody fragments were analyzed with enzyme-linked immunosorbent assay (ELISA) and the uptake and retention of the 125I labeled antibody fragments were evaluated using HeLa HEp-2 multicellular tumor spheroids (MCTSs). Analysis of the antibody fragments demonstrated that parts of the α-factor remained at the N-terminal of the antibody fragments. Despite incomplete processing of the α-factor, the antibody fragments were functional where the sc(Fv)2s gave a three-fold stronger signal in ELISA compared to their scFv counterparts and the mutant antibodies demonstrated a stronger signal than their initial wild types. In addition, the sc(Fv)2s DiTS1-218 and DiHE1-Q displayed an approximately two-fold higher uptake and were retained to a larger extent in the MCTS, demonstrating a 3.9 and 9.4-fold increase in half-life respectively compared to their corresponding scFvs. In conclusion, expression in P. pastoris improved the yield 20-fold and facilitated the purification of the antibody fragments. Furthermore, the sc(Fv)2s presented a higher functional affinity to K 8 both in ELISA and MCTS compared to the scFvs with DiHE1-Q being the best candidate for further studies.
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| 40. |
- Jahnmatz, Peter, et al.
(författare)
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An antigen-specific, four-color, B-cell FluoroSpot assay utilizing tagged antigens for detection
- 2016
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 433, s. 23-30
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Tidskriftsartikel (refereegranskat)abstract
- The FluoroSpot assay, a variant of ELISpot utilizing fluorescent detection, has so far been used primarily for assessment of T cells, where simultaneous detection of several cytokines has allowed a more qualitative analysis of functionally distinct T cells. The potential to measure multiple analytes also presents several advantages when analyzing B cells. Our aim was to develop a B-cell FluoroSpot assay adaptable to studies of a variety of antigens. The assay utilizes anti-IgG antibodies immobilized in 96-well filter membrane plates. During cell culture, IgG antibodies secreted by antibody-secreting cells (ASCs) are captured in the vicinity of each of these cells and the specificity of single ASCs is defined using antigens for detection. The antigens were labeled with biotin or peptide tags enabling secondary detection with fluorophore-conjugated streptavidin or tag-specific antibodies. The assay, utilizing up to four different tag systems and fluorophores simultaneously, was evaluated using hybridomas and immunized splenocytes as ASCs. Assay variants were developed that could: i) identify multiple ASCs with different antigen specificities; ii) detect ASCs showing cross-reactivity with different but related antigens; and iii) define the antigen-specificity and, by including anti-IgG subclass detection reagents, simultaneously determine the IgG subclass of antibodies secreted by ASCs. As demonstrated here, the B-cell FluoroSpot assay using tag-based detection systems provides a versatile and powerful tool to investigate antibody responses by individual cells that can be readily adapted to studies of a variety of antigen-specific ASCs.
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| 41. |
- Jahnmatz, Peter, et al.
(författare)
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Multiplex analysis of antigen-specific memory B cells in humans using reversed B-cell FluoroSpot
- 2020
-
Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 478
-
Tidskriftsartikel (refereegranskat)abstract
- Analysis of B-cell specificities at the single cell level provides important information on how the B-cell compartment responds when challenged by infection or vaccination. We recently developed a reversed B-cell FluoroSpot assay and showed that it could be used to detect B cells specific for different antigens simultaneously in a mouse model. The aim of this study was to further develop the method to detect and quantify antigen-specific memory B cells (MBCs) in humans where circulating antigen-specific cells are less frequent. We show that MBCs specific for three antigens, tetanus toxoid, hepatitis B surface antigen and cytomegalovirus pp65, could be detected simultaneously in one well. In addition to enumerating antigen-specific MBCs, we also assessed the spot volume to estimate the intensity of the response in individual cells and found this to be a new and sensitive approach to study MBC responses after vaccination. This unique B-cell FluoroSpot approach provides a simple and sensitive multiplex analysis of MBCs and can be adapted to most antigens and host species.
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| 42. |
- Johansson, Daniel X, et al.
(författare)
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Efficient expression of recombinant human monoclonal antibodies in Drosophila S2 cells
- 2007
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 318:1-2, s. 37-46
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Tidskriftsartikel (refereegranskat)abstract
- We have explored the Drosophila S2 cell line for expression of Ig molecules isolated as Fab or scFv cDNA from phage-displayed libraries. We present a series of vectors for inducible expression and secretion of human Ig heavy (HC) and light chains (LC), both on separate plasmids and in combination constructs. Both HC (tested as human γ1) and LC (human κ) could be expressed separately and were secreted into the medium, confirming previous reports. When the combination vector carrying both the HC and LC cDNA, as well as when the HC and LC vectors were co-transfected, complete IgG1 was found in the medium. Transient transfection resulted in production levels of 0.5-1 mg/l. Stable cell lines could be established within 2-3 weeks. After 10-12 days of expression from such cell lines, Ig molecules accumulated and the medium contained typically 5-35 mg/l of IgG1. The IgG in these preparations was purified to more than 90% purity on protein G columns. Binding characteristics for IgG of the same clone expressed in S2 cells or mammalian cells were indistinguishable. The main advantages with this system compared to mammalian expression were its robustness and the much faster establishment of stable, high level producing cell lines. © 2006 Elsevier B.V. All rights reserved.
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| 43. |
- Johansson, Ewa, 1964, et al.
(författare)
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Development of monoclonal antibodies for detection of Antisecretory Factor activity in human plasma.
- 2009
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Ingår i: Journal of immunological methods. - : Elsevier BV. - 1872-7905 .- 0022-1759. ; 342:1-2, s. 64-70
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Tidskriftsartikel (refereegranskat)abstract
- Antisecretory Factor (AF) is expressed in most tissues and can be demonstrated in plasma and other body fluids. Most of the AF in plasma is in an inactive form and activation of AF occurs after exposure to bacterial toxins or after intake of various dietary components. Patients with chronic diseases involving disturbances in inflammatory and secretory processes may benefit from an AF-inducing diet. The aim of the present study was to develop an in vitro assay for the analysis of AF-activity in human plasma. Monoclonal antibodies were raised against a native form of AF prepared from human placenta. Nine clones of the monoclonal antibodies recognizing AF and AF peptides were identified. With the aid of these antibodies, we developed a sensitive ELISA method for direct detection of AF-activity in human plasma. The AF activity in plasma from five healthy volunteers was low, 0.112+/-0.022 (absorbance at 405 nm), before intake of the AF-inducing diet with the SPC-Flakes, and increased significantly (p<0.05) to 0.444+/-0.068 after >or=6 weeks on the diet. A comparison of the plasma-AF values, obtained by the bioassay and the immunogenic assay (indirect ELISA), shows that there is a significant correlation (r=0.85) between the values from the two methods. The results indicate that the ELISA measures AF-activity and has the potential to be an important tool for the analysis of AF-activity in further clinical studies on AF-therapy.
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| 44. |
- Johansson, Jessika, 1990, et al.
(författare)
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Phagocyte interactions with Mycobacterium tuberculosis - Simultaneous analysis of phagocytosis, phagosome maturation and intracellular replication by imaging flow cytometry.
- 2015
-
Ingår i: Journal of immunological methods. - : Elsevier BV. - 1872-7905 .- 0022-1759. ; 427, s. 73-84
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Tidskriftsartikel (refereegranskat)abstract
- Utilization of compounds that enhance the innate immune response against Mycobacterium tuberculosis is an attractive strategy for combating tuberculosis in the post-antibiotic era. Thus, it is crucial to develop methods that can be used to screen for such compounds and to investigate their mechanisms of action. Here, we used imaging flow cytometry (ImageStreamX Mk II), which enables rapid quantification of microscopic images in flow, to study the interaction between phagocytes and M. tuberculosis. Macrophage-differentiated THP-1 cells were infected with GFP-expressing M. tuberculosis H37Ra, and methods for rapidly assessing phagocytosis, phagosome maturation, and bacterial replication inside the cells were developed and evaluated. These aspects of innate immunity are essential in determining the outcome of mycobacterial infection of phagocytes. The technique was found effective for monitoring phagocytosis of mycobacteria, phagosomal acidification and phagolysosomal fusion, as well as for measuring mycobacterial replication inside the cells. Several of these aspects could be analyzed simultaneously in the same sample, providing a great deal of information about the phagocyte–mycobacterial interaction at once. Thus, this method has great potential to be useful both for basic research questions and for evaluating compounds that enhance the innate immune response against M. tuberculosis.
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| 45. |
- Johansson, R, et al.
(författare)
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Transiently binding antibody fragments against Lewis x and sialyl-Lewis x
- 2006
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 1872-7905 .- 0022-1759. ; 312:1-2, s. 20-26
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Tidskriftsartikel (refereegranskat)abstract
- Biomolecular recognition is often characterised by low affinity where many weak interactions work either alone or in concert, resulting in an inherent dynamic situation. For example the well-studied weak binding of cell-cell interactions is predominantly based on a range of carbohydrates that interact with numerous (protein) ligands. Finding appropriate binders to these carbohydrate structures may pave the way for new analytical strategies based on low affinity, and recombinant antibody technology is a promising approach to the development of such reagents. We have in the present study characterised two low affinity human single chain antibody fragments (scFv) by surface plasmon resonance for use in such applications. The two clones, LeX1 and sLeX10, had been selected from a naive phage display library against Lewis x (Le(x)) and sialyl Le(x) (sLe(x)), respectively. Both LeX1 and sLeX10 showed low affinity, with K-D values of 3.5 +/- 0.7 x 10(-5) M for Le(x) and 2.6 +/- 0.7 x 10(-5) M for sLe(x), respectively. Kinetic studies revealed the scFvs to be associated with fast dissociation rates, with K-d values higher than 0.1 s(-1) for both LeX1 and sLeX10. Apart from the Lewis structures Le(x) and sLe(x), we investigated the conforinational isomers Lewis a and sialyl-Lewis a together with the monosaccharide units of the Lewis structures, and both scFvs showed high specificity for their respective carbohydrate. Taking these observations together we have demonstrated that scFv with fast reaction kinetics and low affinity have the necessary characteristics for further development as specific tools in analytical strategies, e.g. differentiation of cells based on the various configurations of carbohydrate epitopes. (c) 2006 Elsevier B.V. All rights reserved.
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| 46. |
- Karlsson, Jennie, 1979, et al.
(författare)
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A methodological approach to studies of desensitization of the formyl peptide receptor: Role of the read out system, reactive oxygen species and the specific agonist used to trigger neutrophils.
- 2010
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Ingår i: Journal of immunological methods. - : Elsevier BV. - 1872-7905 .- 0022-1759. ; 352:1-2, s. 45-53
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Tidskriftsartikel (refereegranskat)abstract
- Neutrophil accumulation at an inflammatory site or an infected tissue is dependent on the recognition of chemotactic peptides that bind to G-protein coupled receptors (GPCRs) exposed on the surface of the inflammatory cells. A GPCR activated by a chemoattractant quickly becomes refractory to further stimulation by ligands using the same receptor. This desensitization phenomenon has been used frequently to characterize new receptor agonists and to determine receptor hierarchies. In this study we show that desensitization patterns differ depending on what read out systems are used to follow neutrophil activity. When monitoring release of superoxide, neutrophils were readily desensitized against repeated stimulations with the prototypical agonist formylmethionyl-leucyl-phenylalanine (fMLF). In contrast, neutrophils were not desensitized for fMLF when cell activity was determined by intracellular calcium ([Ca(2+)](i)). The difference observed was dependent on inactivation of the agonist in one read out system but not in the other, and we suggest several different solutions to the problem. Agonist inactivation occurs through a myeloperoxidase (MPO)/hydrogen peroxide catalyzed reaction, and the problem could be avoided by using a FACS based technique to measure the change in [Ca(2+)](i), by the use of an agonist insensitive to the MPO/hydrogen peroxide-system or, by adding an MPO inhibitor or a scavenger that removes either superoxide/hydrogen peroxide or the MPO-derived metabolites.
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| 47. |
- Ketelsen, Anna, et al.
(författare)
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SENSITIVE DETECTION OF HYDROPHOBIC ANTIGENS USING A NOVEL LIPID-AGGREGATE BASED ELISA
- 2008
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; :2008
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies against hydrophobic antigens are common in several autoimmune diseases. However, detection of such antibodies by standard immune-assays, such as ELISA, is problematic, in part because of the problems with coating hydrophobic molecules onto polystyrene multi-well plates. We describe a novel method of stably associating hydrophobic antigens to ELISA plates. By mixing the antigen with a hydrophobic molecule containing a hydrophilic anchor, we generate mixed lipid aggregates that can attach to ELISA plates, and are resistant to detergent wash. Using the ganglioside GM-1 and phosphatidylethanolamine conjugated to the hapten DNP (dinitrophenyl) as model antigens, we show that hydrophobic antigens incorporated into mixed lipid aggregates expose their antigenic determinants in a correct configuration. The detection limit of both GM-1 and DNP-PE was considerably improved compared to when these antigens were coated on ELISA plates using organic solvents. Furthermore GM-1 incorporated into mixed lipid aggregates can be detected by specific antibodies in patient serum. The method of incorporating hydrophobic antigens into mixed lipid aggregates for stable association to ELISA plates can presumably be applied to a vast array of hydrophobic antigens, and may well be developed into a large scale screening system for serum reactivity towards different hydrophobic antigens.
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| 48. |
- Kivling, Anna, et al.
(författare)
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How and when to pick up the best signals from markers associated with T-regulatory cells?
- 2009
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 345:1-2, s. 29-39
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Tidskriftsartikel (refereegranskat)abstract
- Regulatory T (Treg) cells are important tools with the purpose to control and regulate the immune system. These cells use FOXP3, TGF-beta, CTLA-4 and sCTLA-4 to regulate other T-cells. Since cryopreservation of peripheral blood mononuclear cells (PBMC) is a convenient way to handle samples, we investigated whether these cells will change their mRNA expression of Treg associated markers, as well as secretion of TGF-beta1, after cryopreservation. Additionally, we aimed to investigate the mRNA expression after two different time intervals of in vitro antigen stimulation. PBMC from healthy adults were stimulated either fresh (48/96 h) or after cryopreservation (48 h), with PHA, tetanus toxoid, beta-lactoglobulin, ovalbumin or in culture medium exclusively (spontaneous). The mRNA expression of FOXP3, TGF-beta, CTLA-4 and sCTLA-4 were studied with multiplex real-time RT-PCR and TGF-beta1 with ELISA. Cryopreserved PBMC were appropriate for detection of the Treg associated markers FOXP3, TGF-beta, CTLA-4 and sCTLA-4 expressed spontaneously. Also antigen-induced mRNA expression of CTLA-4, sCTLA-4 and TGF-beta and secreted TGF-beta1, with the exception of FOXP3, preserved a stable transcription activity after cryopreservation. Further, a stimulation period of 48 h in general revealed the highest mRNA expression of the markers studied. In conclusion, cryopreserved cells are in general suitable for studying Treg associated markers.
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| 49. |
- Kumar, Ashok, et al.
(författare)
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Affinity fractionation of lymphocytes using a monolithic cryogel.
- 2003
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 1872-7905 .- 0022-1759. ; 283:1-2, s. 185-194
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Tidskriftsartikel (refereegranskat)abstract
- A new type of continuous, supermacroporous, monolithic, cryogel affinity adsorbent was developed, allowing specific fractionation and separation of human peripheral blood lymphocytes in a chromatographic format. The affinity adsorbent was used to design a novel cell separation strategy, which was based on the interaction of protein A from Staphylococcus aureus with cells bearing IgG antibodies on the surface. After treating lymphocytes with goat anti-human IgG(H+L), the IgG-positive B-lymphocytes were efficiently separated from T-lymphocytes. Protein A covalently coupled to epoxy activated dimethylacrylamide (DMAA) cryogel matrix specifically bound IgG-bearing B-lymphocytes through the Fc region, while non-bound T-lymphocytes passed through the column. More than 90% of the B-lymphocytes were retained in the column while the cells in the breakthrough fraction were enriched in T-lymphocytes (81%). The viability of the T-lymphocytes isolated was greater than 90%. The bound lymphocytes released by human or dog IgG recovered 60–70% of the B-cells without significantly impairing the cell viability. The technique can be applied in general to cell separation systems where IgG antibodies against specific cell surface markers are available.
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| 50. |
- Larsson, Anders, et al.
(författare)
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The effects of age and gender on plasma levels of 63 cytokines
- 2015
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 425, s. 58-61
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Tidskriftsartikel (refereegranskat)abstract
- Cytokines play important roles as regulators of cell functions, and over the last decades a number of cytokine assays have been developed. The aim of the present study was to investigate the effects of age and gender on a large number of cytokines. Plasma samples were collected from 33 healthy blood donors. The samples were analyzed using the multiplex proximity extension assay (PEA) allowing simultaneous measurement of 92 cytokines and four technical controls. Biomarkers with less than 80% quantitative results were excluded leaving 63 cytokines that were analyzed for the effects of gender and age. The plasma level of three of the investigated biomarkers (DNER, MCP-4 and MMP-10) were found to be significantly different for the two genders (adjusted p-value <0.05), and 15 of the biomarkers (CCL11, CCL25, CDCP1, CSF-1, CXCL11, CXCL9, FGF-23, Flt3L, HGF, IL-10RB, MCP-3, MCP-4, MMP-10, OPG, VEGF-A) were significantly associated with age. This study reveals the effects of age and gender on a large number of cytokine assays. CXCL5 and TNFB were significantly higher in females, while the other markers with significant gender-dependent differences were higher in males. For the markers that were significantly associated with age, only CXCL6 was found to decrease with age, while the other biomarkers increased with age.
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