Radial immunodiffusion

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. 

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

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. 

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). 

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

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

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). 

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. 

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

B3. Berner, J. J., Ciemins, V. A., and Schroeder, E. R, Radial immunodiffusion of spinal fluid Diagnostic value in multiple sclerosis. AJCP 58, 145-152 (1972). 

Radial immunodiffusion procedures are varied but all depend upon the diffusion of the antigen or antibody in a gel producing a precipitate which is proportional to the quantity of reactants (often sensitive to 25 ng protein with visual methods (32,72, 73)). Modifications using radiolabeled antigens or antibodies may increase the sensitivity fifty-fold (74,75). If the antibody is first mixed with the hapten, the concentration of free unbound antibody will decrease proportionately and result in a decrease in the precipitate formed with the antigen which can be observed visually or with radiolabeled methods. 

Cost Effectiveness. As with the other advantages of immunochemical analysis, cost may be quite variable. Reagent costs for several automated systems have been estimated at under 1.25 per sample. The cost is obviously much lower for less sophisticated assay systems, especially if some reagents are prepared in house. A major consideration is the expense of new instrumentation. For dedicated or automated instrumentation for either RIA or ELISA procedures, the cost may be 50-100,000. However, most analytical laboratories already have the basic instrumentation needed for immunoassays. Moderate sensitivity can be obtained through the use of numerous procedures such as radial immunodiffusion and hemagglutination. These procedures require no expensive equipment or reagents and they may be very useful in areas where equipment acquisition or maintenance is a problem. 

Although it is possible to estimate apoC concentrations in lipoprotein fractions by delipidation, electrophoresis or isoelectric focusing, staining, and densitometry [e.g., (C5, N3)], or by high-pressure liquid chromatography (H6), most reported measurements of plasma apoC concentration have been by immunological means. These include radioimmunoassay (K8, K9, S17), electroimmunoassay (A4, A5, C27), radial immunodiffusion (P21), and enzyme immunoassay (H29). 

The concentration of apoD in human serum has been reported as 10-12 mg/dl by electroimmunoassay and radial immunodiffusion (C25) and a little less (6.2 1.0 mg/dl for males, 5.6 1.4 mg/dl for females) by radial immunodiffusion (A 14). In the latter study, apoD was significantly correlated with HDL cholesterol levels in both sexes (A14). 

C25. Curry, M. D., McConathy, W. J., and Alaupovic, P., Quantitative determination of human apolipoprotein D by electroimmunoassay and radial immunodiffusion. Biochim. Biophys. Acta 491, 232-241 (1977). 

It should also be emphasized that although the precision of the automated particle-enhanced immunometric methods is higher than that of the enzyme-amplified single radial immunodiffusion first used to quantitate serum cystatin C, it is still lower than that for most methods for determination of serum creatinine. Moreover, the data for the intraindividual variation of serum cystatin C strongly indicate that a higher precision of the method would markedly improve the clinical usefulness of serum cystatin C determinations according to the criterion of Cotlove (N6). 

Fig. 15. Temperature stability of wild-type cystatin C and of L68Q-cystatin C, the cerebral hemorrhage-producing cystatin C variant. Samples of L68Q- and wild-type (wt) cystatin C were incubated for 30 min at various temperatures. (A) shows agarose gel electrophoresis at pH 8.6 of selected samples. The point of application and the anode are marked by an arrow and a plus sign, respectively. (B) shows die remaining cystatin C-immunoreactivity of sample supernatants after incubation and centrifugation as determined by single radial immunodiffusion.

Figure 6-3. Radial immunodiffusion. Antigen solutions at various concentrations are loaded into wells cut into agar gels containing a fixed concentration of antibody (left). A precipitin ring...

Radial immunodiffusion and double diffusion experiments can be accelerated by electrophoresis. In rocket electrophoresis, so-called because of the shape of the zone of precipitation, electrophoresis of proteins is carried out in an agarose gel which contains antibody. The precipitated antibody-antigen complexes form rocketshaped zones when the pH is adjusted (often pH 8.0) so that antigen and antibody migrate in opposite directions (Figure 6-6)... 

Mancini, G., Carbonara, A. O, and Herenians,J. F. (1965) bninunochetiucal quan utauon of antigens by single radial immunodiffusion. Immunochemtstry 2, 235-254. 

Blanchard, G.C. Gardner, R. Two immunofluorescent methods compared with a radial immunodiffusion method for measurement of serum immunoglobuhns. Clin. Chem. 1978, 24, 808-814. 

O Brien TC, Maloney CJ, Tauraso NM. Quantitation of residual host protein in chicken embryo-derived vaccines by radial immunodiffusion. Appl Microbiol 1971 21(4) 780-2. 

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