Immunodiffusion

Depending on the question, fill the central well either with the antigen solution or with antiserum. Place the antigen(s) or antisera or antiserum dilutions into the peripheral wells. If the wells are not filled completely, adjust with PBS. 

Place the slides horizontally in a humid chamber, e.g., Petri dishes containing wet filter paper, and store in a refrigerator at least for 24 h, better for 48-72 h. 

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Immunodiffusion and immunoprecipitation, developed ia the 1940s as a means to identify and semiquantitate specific proteias, were the direct precursors to the development ia 1953 of Immunoelectrophoresis, a method used ia many clinical laboratories (5). Single- and double-gel immunodiffusion and immunoelectrophoresis were, ia effect, the first standardized and routinely used immunoassay methods (see Electroseparations, electrophoresis). 

Standardization and Testing". The final vaccine is tested for safety, potency, and residual chemicals. Safety includes testing for endotoxin and stetihty. Potency is evaluated by quantitative determination of the amount of hemagglutinin in the vaccine. Antibody to this glycoprotein is associated with protection. The single radial immunodiffusion (SKID) technique is used to standardi2e the mass of this protein in comparison to a reference preparation. 

Immunoelectrophoretic Techniques. The technique of gel electrophoresis has been successfully combined with immunological techniques in order to further evaluate molecules. Specifically, the concept of double immunodiffusion as described in 1948 (57) and that of single-radial immunodiffusion described in 1963 (58) have been further developed for use with electrophoresis in both the clinical and research setting. 

The double-immunodiffusion technique, often referred to as the Ouchtedony technique, uses an agarose gel as the matrix. Holes are made in the agarose where either sample or antisera is placed. The two solutions are allowed to diffuse into the matrix for a predetermined time. If there is a reaction... 

Mancini G, Carbonara AO, Heremans JE. 1965. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochem 2 235-254. 

Influenza (split virion)t Allantoic fluid from embryonated hens eggs infected with influenza viruses A and B 1 Harvest of viruses 2 Disruption with surface active agent 3 Blending of components of different serotypes Assay of haemagglutinin content by immunodiffusion Inoculation of embryonated hens eggs to exclude live virus... 

In most patients, serologic evidence remains the primary method in the diagnosis of histoplasmosis. Results obtained from complement fixation, immunodiffusion, and latex antigen agglutination antibody tests are used... 

Taylor, B.C. et al., Measurement of serum immunoglobin concentration in killer whales and sea otters by radial immunodiffusion, Vet. Immunol. Immunopathol., 89, 187, 2002. 

Double helixes, self-recognition in the self- assembly of, 16 803 Double-heterojunction (DH) structures, for LEDs, 22 173, 174, 175 Double heterostructure (DH), 14 844 Double heterostructure laser diodes, 14 700 Double hetero structure OLEDs, 22 216 Double-immunodiffusion technique, 9 753-754... 

Immunocytochemical staining methods, for flax fiber, 11 599 Immunodiffusion, 14 136 Immunoelectrophoresis, 14 136... 

Single-point fuel injection system, 10 51 Single-product batch processes, 20 723 Single radial immunodiffusion (SRID) technique, 25 494... 

The D antigen unit is another form of strength expression of viral vaccines. The D antigen units are determined on the basis of radial immunodiffusion (e.g., poliovirus vaccine). 

Kerenyi L, Galiyas F (1972) A highly sensitive method for demonstrating proteins in electrophoretic, immunoelectrophoretic and immunodiffusion preparations. Clin Chim Acta 38 465 167... 

Single radial immunodiffusion (SRID) is a quantitative immunoprecipitation technique. 

Procedure 7.1 Quantitation of albumin by single radial immunodiffusion... 

Double immunodiffusion techniques permit the quantitation of one protein in the presence of other proteins... 

The discovery of Lp(a) by Berg in 1962 (B6) relied on the production of rabbit antisera against beta-lipoprotein and on the selective absorption of these antisera with individual human sera. When certain human sera were used for absorption, the antisera retained precipitation capacity in radial immunodiffusion with 30-35% of individual human sera, which obviously contained a previous unknown antigen. The particle carrying the new antigen shared antigenic properties with beta-lipoprotein, but had an additional antigenic structure. This was evidenced from the only partial fusion of the precipitin bands formed between a positive human serum, the antibeta lipoprotein antiserum and the new absorbed antiserum. 

Electroimmunoassay (rocket electrophoresis) and radial immunodiffusion (A5) lack sensitivity at low Lp(a) concentrations, and the response is influenced by the size of the apo(a) isoforms (A5, K28). Differences in migration velocity in the agarose gel lead to an underestimation of the samples with large apo(a) isoforms and to an overestimation of samples with small apo(a) isoforms. Moreover, the detection limit lies around 0.07-0.08 g/liter Lp(a), so that this method is better suited for screening and detection of individuals with elevated Lp(a) levels than for the exact measurement of the plasma Lp(a) concentration. 

C9. Craig, W. Y., Poulin, S. E., Forster, N. R., Neveux, L. M., Wald, N. J., and Ledue, T. B., Effect of sample storage on the assay of lipoprotein(a) by commercial available radial immunodiffusion and enzyme-linked immunosorbent assay kits. Clin. Chem. (Winston-Salem, NC) 38, 550-553 (1992). 

Another indicator that the SEs share similar structures is evidenced by cross-reactivity and neutralization with antibodies. Several years ago, when there were less SEs known, these molecules were considered serologically distinct entities as determined by antisera and relatively insensitive immunodiffusion assays. However, subsequent studies employing a more sensitive technique (ELISA) with polyclonal and monoclonal antibodies clearly reveal that common epitopes do indeed exist between these toxins. 

Quantitation of Human IgG. Radial immunodiffusion technique (Tri-Partigen Calbiochem-Behring Corp.) was used to measure normal serum and serum from byssinotic persons. After filling the wells, diffusion was allowed to proceed for 50 hr at 4°C. Precipitin rings were measured and the concentration of IgG in mg/dl was obtained from the Table of References supplied with the plates. 

Figure 7. Immunodiffusion of normai and byssinotic blood sera against human IgG antibodies. Tripatigen plate contains antibodies to human IgG in gel. Key well 1, control IgG serum provided with plates well 2, normal human serum well 3, serum from byssinosis patient.

The classical methods of demonstrating specificity of antibody reactions with antigens, such as immunodiffusion plates, are obsolete. Immunofluorescence is a very sensitive method that requires controls for specificity of equal sensitivity. The most important controls are not just those that demonstrate the presence of an antibody that reacts with the antigen of interest, but more importantly, the absence of antibodies that react with other things. 

W2. Werner, M., Estimation of blood lipoproteins by radial immunodiffusion after agarose gel filtration. J. Chromatogr. 28, 59-68 (1967). 

LI. Lamy, T., Dosage des immunoglobulines s5riques au cours de la trypanosomiase Africaine par immunodiffusion simple. Thbse Doctoral, University de Dakar, 1966. 

The titre of antibodies in laccase antisera was 1 16 as measured by the immunodiffusion technique of Ouchterlony (21). These antibodies were effective inhibitors of catechol oxidase activity, with 100/il of antisera reducing activity by 95%, in an assay mixture of 3ml of 0.1M catechol in... 

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