| 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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