A new endpoint for ELISA titrations
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Slide 1 :
A new end-point for ELISA titrations Author: José Vidal Related publication: J. Vidal. (2004). A new end-point for ELISA titrations. J. Immunoassay & Immunochemistry, 25 (4), 371-383.
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Background.Enzyme-linked immunosorbent assay (ELISA) is widely used to measure antibody concentrations. Antibody concentration in a sample is determined by comparing the absorbances corresponding to a serial dilution of the sample with the absorbances corresponding to a reference sample. There are two problems:(i) there is no universal curve that fits the points in any ELISA (universal curve for all the ELISAs)(Plikaytis & Carlone, 2005), and
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Background (continued).(ii) sometimes, the sample curve and the reference curve are not parallel (therefore, no precise measurement of the antibody content in the sample is possible).
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Objective.The present communication aims at providing an alternative to curve-fitting. This alternative is based on the color change that occurs in a serial dilution of an antibody sample: toward the end of the dilution, a sudden increase in color is followed by a gradual decrease toward background color; that color increase constitutes an end-point.The antibody presence is revealed with peroxidase-diaminobenzidine-hydrogen peroxide, and further color intensification with silver. (Ludany et al., 1993.)
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Methods.* ?-chain-specific antibodies to human IgG (from goat, Sigma) were bound to ELISA plates (Costar) by poly(L-lysine, phenylalanine) (F.W. 20000-50000, Sigma) and glutaraldehyde (Hobbs, 1989). It also works with antibodies adsorbed on the plastic and fixed with glutaraldehyde. * Samples containing human IgG were serially diluted in PBS containing 0.05% Tween 20 and 5 mg/ml gelatin. * After 1.5-2 hr, peroxidase-bound goat antibodies to human ? and ? chains were added in PBS-Tween-gelatin (the optimal dilution had to be determined previously).
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Methods (continued).* After 2 hr, the presence of antibodies was revealed by addition of 3,3-diaminobenzidine (0.2 mg/ml), nickel chloride (0.5 mg/ml), and H202 (0.002%) in 50 mM Tris-HCl buffer, pH 7.6. (A H202- urea adduct may be used as a source of H202.)* After 20-30 min, the ensuing color was intensified by reaction with a silver reagent (Ludany et al., 1993; Merchenthaler et al., 1989) for 15-20 min.
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Methods (continued) * The silver reagent was prepared using 1 ml of ascorbic acid (2 mg/ml), 1 ml of sodium tungstate (50 mg/ml), and 8 ml of this mixture: sodium acetate (160 mg/ml), acetic acid (7 µl/ml), silver nitrate (0.125 ml of 1% silver nitrate/ml), cetylpyridinium chloride (0.0125 ml of 1% cetylpyridinium chloride/ml), Triton X-100 (0.075 ml of 1% Triton/ml).
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Results. The picture (next slide) shows a plate developed as described above.Row B: a sample of human IgG diluted from 1:10000 (B1) to 1:20480000 (B12).Row E: another sample thereof diluted from 1:10000 (E1) to 1:20480000 (E12).Rows A, C, D, F, G, H: negative controls (e.g., some rows contain goat albumin instead of IgG, anti-IgG antibody was not adsorbed on some wells, etc.).Sample B shows a sharp increase in color at dilution 1:80000 (B4), while sample E shows the increase at dilution 1:160000 (E5). Therefore, the IgG content of sample E is twice the content of sample B.
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Conclusion and caveats.This ELISA allows a simple estimation of the antibody content in a sample, relative to the content in another sample. However,* the increase in color is not always as sharp as shown in the above picture (e.g., color in well E4 may be darker than color in well E3, but lighter than color in well E5); therefore, the IgG content of sample E, relative to that of sample B, may be from 1 to 2,* the dilution factor in the above picture was 2; one may decrease the factor (for instance to 1.5) to increase the precision of the measurements, yet, the number of necessary wells increases, and so does the work.Thus, this ELISA provides an approximate estimation of the relative antibody content in a sample.
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References.Hobbs, R.N. (1989). Solid-phase immunoassay of serum antibodies to peptides. Covalent antigen binding to adsorbed phenyalalanine –lysine copolymers. J.Immunol.Methods, 117, 257-266.Ludány, A., Gallyas, F., Gaszner, B., Andrásfalvy, B., Szücs, G., and Kellermayer, M. (1993). Skimmed-milk blocking improves silver post-intensification of peroxidase-diaminobenzidine staining on nitrocellulose membrane in immunoblotting. Electrophoresis, 14, 78-80.
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References (continued).Merchenthaler, I., Stankovics, J. and Gallyas, F. (1989). A highly sensitive one-step method for silver intensification of the nickel-diaminobenzidine end product of peroxidase reaction. J.Histochem.Cytochem., 37, 1563-1565.Plikaytis, B.D., Carlone, G.M. (2005). Statistical considerations for vaccine immunogenicity trials. Part 1: introduction and bioassay design and analysis. Vaccine, 23, 1596-1605.
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