| 1. |
- Abdel-Motal, Ussama M., et al.
(författare)
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Major histocompatibility complex class I binding glycopeptides for the estimation of 'empty' class I molecules
- 1995
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 0022-1759. ; 188:1, s. 21-31
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Tidskriftsartikel (refereegranskat)abstract
- Different forms of major histocompatibility complex (MHC) class I heavy chains are known to be expressed on the cell surface, including molecules which are functionally 'empty'. Direct peptide binding to cells is obvious during sensitization of target cells in vitro for cytotoxic T lymphocyte killing and 'empty' MHC-I molecules are comparatively abundant on TAP- 1 2 peptide transporter mutant cells. In the present work we have estimated the fraction of 'empty' MHC class I molecules using glycosylated peptides and cellular staining with carbohydrate specific monoclonal antibodies. Synthetic Db and Kb binding peptides were coupled at different positions with different di- or trisaccharides, using different spacing between the carbohydrate and the peptide backbone. Binding of sugar specific mAbs was compared in ELISA and cellular assays. An optimal Db binding glycopeptide was used for comparative staining with anti-Db and anti-carbohydrate monoclonal antibodies to estimate fractions of 'empty' molecules on different T lymphoid cells. On activated normal T cells, a large fraction of Db molecules were found to be 'empty'. The functional cole of such 'empty' MHC class I molecules on T cells is presently unclear. However, on antigen presenting cells they might participate in the antigen presentation process.
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| 2. |
- Akerström, B, et al.
(författare)
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On the interaction between single chain Fv antibodies and bacterial immunoglobulin-binding proteins
- 1994
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Ingår i: Journal of Immunological Methods. - 0022-1759. ; 177:1-2, s. 63-151
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Tidskriftsartikel (refereegranskat)abstract
- Using four bacterial immunoglobulin-binding proteins, we have analyzed the binding characteristics of a panel of 34 human single chain Fv antibodies, expressed in E. coli and with known specificity and sequence. Several of the single chain Fv antibodies showed affinity for staphylococcal protein A and peptostreptococcal protein L, but not for the streptococcal proteins G or H. The affinity of the binding was higher for protein L (4.5 and 1.4 x 10(9) M-1) than for protein A (7.7 and 6.7 x 10(8) M-1), using the two single chain Fv antibodies displaying the strongest binding activity to these ligands. The binding was shown to be specific by Western blotting, and the single chain Fv antibodies could be purified from crude bacterial culture media by affinity chromatography on protein L- or A-Sepharose. Protein A, which has affinity for the VH domain of the scFv antibodies, was tested against scFv antibodies containing VH1, VH3, VH4 and VH5 domains, and its binding was restricted to approximately half of the scFv antibodies with a VH3 domain. Protein L, which has affinity for the VL domain, was tested against kappa 1, kappa 4, lambda 1, lambda 2 and lambda 3 domains, and it bound all kappa 1 domains, one lambda 2 and one lambda 3 domain. Comparison of the amino acid sequences of binding and non-binding VL domains demonstrated that amino acid residues crucial to the binding of protein L were distributed over a large area outside the hypervariable antigen-binding regions.
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| 3. |
- Ankerst, J., et al.
(författare)
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Microcalorimetry as a tool for the detection of complement-dependent cytotoxicity
- 1985
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 0022-1759. ; 77:2, s. 267-274
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Tidskriftsartikel (refereegranskat)abstract
- Microcalorimetry using a 4-channel static ampoule microcalorimeter of thermopile type has been evaluated as a tool for the detection of complement-dependent cytotoxicity against the surface antigens of living cells. Cytotoxic reactions mediated by a rabbit antiserum against human white blood cells and by 2 different monoclonal antibodies recognizing a melanoma-associated antigen on a human melanoma cell line were studied. The cytotoxic reactions were registered as a decrease of the heat production rate when the cells were exposed to antibodies in the presence of active complement as compared to the heat production rate of the cells exposed to the same antibodies in the presence of inactive complement. This investigation shows that microcalorimetry can be used as a highly sensitive method for the detection of complement-dependent immune reactions, detecting antibody dilutions higher than 10-5. It also indicates that microcalorimetry may become a particularly important technique in the analysis of the kinetics of cytotoxic immune reactions in vitro.
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| 4. |
- Ankerst, J., et al.
(författare)
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Use of microcalorimetry in analysing the kinetics of ADCC
- 1986
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 0022-1759. ; 88:2, s. 259-264
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Tidskriftsartikel (refereegranskat)abstract
- Microcalorimetry was found to be a useful technique for the demonstration of antibody-dependent cellular cytotoxicity (ADCC) against human melanoma cells mediated by a heterologous rabbit antiserum and two monoclonal antibodies in combination with human peripheral blood lymphocytes as effector cells. The rabbit antiserum and the monoclonal IgG3 antibody 2B2 directed against the GD3 ganglioside expressed cell-inhibitory effects resulting in a decreased heat production rate over 2-18 h of incubation. The 4.2 monoclonal IgM antibody to GD3 had no similar cell-inhibitory effect. In contrast, the 4.2 antibody expressed a much stronger effect than 2B2 in tests for complement-dependent cytotoxicity. The kinetics of these effects were quite reproducible. It is concluded that microcalorimetry is a sensitive and particularly suitable method for the analysis of cytotoxicity kinetics.
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| 5. |
- 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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| 6. |
- Elbashir, M I, et al.
(författare)
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Antibody response in immunized rabbits measured with bacterial immunoglobulin-binding proteins
- 1990
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 0022-1759. ; 135:1-2, s. 9-171
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Tidskriftsartikel (refereegranskat)abstract
- Protein G, an immunoglobulin (Ig)-binding protein isolated from group C or G streptococci, binds to the Fc portion of IgG. Protein L, from the anaerobic bacterium Peptostreptococcus magnus, specifically binds light chains of Ig. In this study, protein G and L were used to measure the production of antibodies in immunized rabbits. Two rabbits were immunized with a mixture of human urinary proteins from a patient with tubular proteinuria, and blood samples were collected regularly from the animals for 6 weeks after the immunization. The antibody levels of the blood samples against six of the proteins in the antigen mixture were then measured by ELISA. Microtiter plates were coated with each of the antigens, incubated with the rabbit serum samples, and the specific antibodies of the IgG class measured by incubation with biotinylated protein G, and antibodies of all Ig classes with biotinylated protein L. Alternatively, Western blotting was employed, where the antibodies which bound to each antigen after separation by SDS-PAGE and transfer to nitrocellulose membranes, were detected by protein G or L. The results showed that antibody production against five of the antigens, albumin, alpha 1 gamma-acid glycoprotein, alpha 1 gamma-microglobulin, Ig light chains, and retinol-binding protein, showed a similar pattern, although the magnitude of the initial IgM response differed somewhat. After 6 weeks, the levels of the protein G-binding antibodies had reached a plateau, while those of protein L-binding antibodies were still increasing. The response to the sixth antigen, beta 2 microglobulin, was considerably different. A dramatic increase of anti-beta 2 gamma-microglobulin antibodies was seen during the 4th week after immunization when protein L was used.
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| 7. |
- Fernandez-Luna, J L, et al.
(författare)
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A sensitive and rapid enzyme-linked immunosorbent assay using monoclonal antibodies for simultaneous quantitation of free and IgA-complexed protein HC
- 1985
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 0022-1759. ; 82:1, s. 101-110
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Tidskriftsartikel (refereegranskat)abstract
- A sensitive and rapid enzyme-linked immunosorbent assay for simultaneous quantitation of free and IgA-complexed human protein HC was developed with monoclonal murine antibodies. The total amount of protein HC (free plus IgA-complexed) was measured by a competitive procedure while the protein HC-IgA complex was quantitated by a sandwich enzyme immunoassay. The amount of free protein HC was then obtained as the difference between the 2 measured values. The sensitivity of the procedure was 70 micrograms/1 for the total amount of protein HC and 80 micrograms/1 for the protein HC-IgA complex. At ordinary human serum levels of free protein HC and protein HC-IgA complexes the coefficient of variation for the procedure was about 5%. One worker could determine the concentrations of free and IgA-complexed protein HC in up to 400 serum samples in a working day.
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| 8. |
- Fredrikson, G, et al.
(författare)
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Use of protein G for preparation and characterization of rabbit antibodies against rat adipose tissue hormone-sensitive lipase
- 1987
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 0022-1759. ; 97:1, s. 65-70
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Tidskriftsartikel (refereegranskat)abstract
- The newly described immunoglobulin G-binding streptococcal surface protein, protein G, was used to prepare and characterize rabbit antibodies. The antibodies were directed against rat hormone-sensitive lipase, the rate-limiting enzyme in the hydrolysis of the triacylglycerols stored in adipose tissue. Antiserum was obtained after two injections with 20 micrograms enzyme protein, and the immunoglobulin fraction was obtained using a protein G-based solid-phase radioimmunoassay. The hydrolysis of acylglycerols by the enzyme was inhibited by the antibodies, and the enzyme could be efficiently removed from a solution using the antibodies and heat-killed streptococci expressing surface protein G. By Western blot and detection with 125I-protein G, the antibodies were found to selectively bind to hormone-sensitive lipase and to a smaller extent to two minor contaminants, possibly proteolytic fragments of the lipase. The amount of 125I-labelled protein G bound to the lipase on the blot was quantitatively related to the amount of enzyme protein down to the detection limit 10 ng.
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| 9. |
- Hardy, Eugenio, et al.
(författare)
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Enhanced ELISA sensitivity using TCA for efficient coating of biologically active lipopolysaccharides or lipid A to the solid phase
- 1994
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 0022-1759. ; 176:1, s. 111-116
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Tidskriftsartikel (refereegranskat)abstract
- A new simple, reproducible and sensitive ELISA that uses trichloroacetic acid (TCA) for the coating of lipooligosaccharide (LOS), smooth lipopolysaccharide (LPS) or lipid A to the solid phase has been developed. The experimental parameters (temperature of coating, time of coating, antigen concentration and TCA concentration) were evaluated by a complete factorial design (24). As a result of the evaluation, two main coating procedures were developed. In one, LOS was shown to coat efficiently in 0.2% TCA, at 37°C, when incubated for only 30 min. In the other procedure, LOS in 0.2% TCA was coated at 37°C for 16 h. The slower procedure proved, as expected, to be even more efficient than the former. The new ELISA was compared to three previously reported ELISAs, and showed the greatest sensitivity, probably, as a consequence of the higher coating efficiency of LOS to plates. The biologic activity of LOS was not modified by the low TCA concentration used, as proven by retention of its biological activity in the induction of procoagulant activity in blood mononuclear cells. We conclude that small amounts of biologically active LOS/LPS or lipid A can be coated on solid surfaces by this approach to achieve a rapid and economical assay procedure.
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| 10. |
- Kjellström, S, et al.
(författare)
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Flow immunochemical bio-recognition detection for the determination of interleukin-10 in cell samples
- 2000
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Ingår i: Journal of Immunological Methods. - 0022-1759. ; 246:1-2, s. 30-119
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Tidskriftsartikel (refereegranskat)abstract
- On- and off-line heterogeneous non-competitive flow immunoassays for the determination of Interleukin-10 are described. The sample containing IL-10 is mixed, either on-line in a reaction coil or off-line in a test tube, with fluorescent labelled anti-IL-10 antibodies to form an antibody-antigen complex. The labelled unbound antibodies are trapped on an immobilized IL-10 column whereas the IL-10-antibody complexes are eluted and detected downstream by a fluorescence detector. The optimization of the systems was performed with respect to choice of affinity support, flow rate, carrier buffer additives, pH and antibody-antigen association. Both bio recognition assays were tested with a spiked cell medium and the IL-10 detection limits in this matrix was found to be 8 fmol using the off-line incubation mode and 40 fmol using the on-line incubation mode. The sample through-put was 26 and 40 samples per hour in the on-line and off-line incubation modes, respectively. IL-10 identification in the sample fractions was achieved using MALDI-TOF MS.
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