Immune precipitation

The potential sequelae to an immune response (formation of neutralizing antibodies, provoking an autoimmune or a hypersensitivity response), and pathology due to immune precipitation, and so on ... 

Chuang further cautions that In solid phase immunoassay, it is generally assumed that antigens adsorbed to a surface, such as polystyrene microtiter dishes, will react with specific antibody in a manner similar to that antigen-antibody reaction in solutions such as occur in immune precipitation. However, our evidence and others89) seem to point out that data obtained from solid phase immunoassays should be interpreted with caution since adsorption of a nonantigen to a polymer surface could render it immunoreactive to previously unreactive antibodies. ... 

The screening assay is, in general, a binding assay, mostly an ELISA-type assay, or a radio-immune-precipitation method. Standard ELISA-type immunoassays are not always considered appropriate, however, for measuring binding... 

Figure 5.5 Inhibition of p34cdc2 kinase by apigenin in colon carcinoma cells. A representative image from the immune precipitation study is shown at the top. Each bar represents the mean SD of three to seven experiments. p < 0.05 vs. vehicle control.. p < 0.05 vs. the vehicle controls. (From Wang et al., Molecular Carcinogenesis, 28 102-110, 2000. With permission.)...

Figure 8-17. Hypothetical structure ot immune precipitates and soluble complexes according to the lattice theory. Numbers refer to mole ratios of antibody to antigen. Dotted lines associated with precipitates indicate that the complexes extend as shown. The precipitates may be visualized as those found in the antibody (Ab) excess zone (A), the equivalence zone (B), and the antigen (Ag) excess zone (C). The soluble complexes correspond to those in supernatants having moderate (D), large ( ), and extreme (F) Ag excess. (From B. D. Davis et al.. Microbiology, 2nd ed., Harper and Row, New York, 1973.)...

Figure 8-38. Inhibition of immune precipitation of I-labeled t>-lactate dehydrogenase by increasing amounts of unlabeled purified D-lactate dehydrogenase (O), a 0,1% Triton X-100 extract of wild type membrane vesicles ( ), a 0.1% Triton X-100 extract of mutant membrane vesicles ( ), and 0.1% Triton X-100 in 0.1 Af potassium phosphate buffer, pH 7.1 (A). The 0.1% Triton X-100 extracts of wild type and mutant vesicles contained the same amounts of protein. The inhibitory effect of the wild type extract was compared with that of a purified i>lactate dehydrogenase preparation containing the same number of units of enzyme activity. [From S. Short and H. R. Kaback, J. Biol. Chent. 250 4285 (1975).]...

As already pointed out, the technique in which the antigens are moved by an electric field into a gel layer containing the antiserum has certain similarities with simple diffusion. Here also, the displacement of the precipitation zone will cease after a finite time that is variable and measured in hours when the antigen has been entirely absorbed in a precipitate with the antibodies. A consequence of the role of the electric field is that it may also entail movement of the antibodies in the gel. It has been pointed out that if antigen and antibodies move in the same direction and at a similar rate, immune precipitation may not occur or bizarre patterns may be seen. ... 

An interesting alternative to NMR investigations for the assignment of the configuration of the cyclic acetal of pyruvated polysaccharides uses quantitative inhibition of specific immune precipitation techniques [25]. Thus, synthetic (R) and (S) 4,6-pyruvated methyl galactosides were applied as inhibitors for the precipitation reaction of Klebsiella polysaccharides with the respective antisera. The (R) isomer was revealed to be a potent inhibitor, and thus proved the K. serotype Kll and K21 polysaccharides to contain 4,6-0-[(R)-l-carboxy-ethylidene]-galactopyranosyl residues. 

Subsequently, antiserum spread directly on the gel causes the protein(s) of interest to precipitate. The immune precipitate is trapped within the gel matrix, and alt other nonprecipi-tated proteins can be removed by washing the gel. The gel may then be stained for identification of the proteins. CRIE is more sensitive than IF in terms of detection limit and also shows better resolution. For instance proteins of closely related or identical electrophoretic mobility can be distinguished better by CRIE, because in IF they will appear as a single band. In practice, IF is technically more efficient than either lEP or CRIE, and it produces patterns that are more easily interpreted. The utility of IF, which is now widely used for the evaluation of myeloma proteins, is illustrated in Figure 9-9. 

This procedure is based on the observation that the immune precipitate, obtained after the addition of POase to an anti-POase antiserum, can be solubilized, after the removal of non-specific proteins... 

Ouchterlony, 0., Interpretation of comparative immune precipitation patterns obtained by diffusion-in-gel techniques. In Immunochemical Approaches to Problems in Microbiology (M. Heidelberger and 0. J. Plescia, eds.), pp. 5-19. Rutgers Univ. Press, New Brunswick, New Jersey, 1960. 

Fig. 2.6 Identification of recombinant clones by direct immunostaining. Colonies were incubated with Texas Red-labeled antibodies against IgC at concentrations allowing the formation of immune precipitates at colonies expressing leptin-Fc...

The strong cross-reaction of the polysaccharide of Aloe vera, - which contains D-mannose, D-glucose, and a small proportion of galactose and a pentose, could also be attributed to the presence of multiple residues of d-glucose, since the gum recovered from the immune precipitate contained no galactose. A portion of the D-glucose in Aloe vera is (l->4)-linked and would account for the observed cross-reaction. 

Determination of the class and subclass of the antibody is performed in a number of ways, including radioimmunoassay, ELISA, and immune precipitation, using subclass-specific antisera. These are commercially available in the form of antibody isotyping kits. 

NaCl, 10 mM MgSO, 0.01% gelatin), precipitated in the presence of PEG and DNA is isolated by phenol-chloroform extraction as described by Maniatis et al. (7). Alternatively, phage from the plate lysate can be obtained by immune precipitation using a polyclonal antibody directed against lambda phage particles (Lambda-Sorb, Promega Biotech). 

Add 1 pL of RNase A/Tl and incubate for 15-30 min at 37 °C. Then add proteinase Kto final concentration ofO.5 mg/ mL and incubate overnight at 37 °C. (This should be done with input DNA from Subheading 3.3, step 2 as well as with the immune precipitates. For this, the input DNA should be brought to a final volume of450 pL with TE buffer.)... 

Numbers represent the amount of radioactivity in the immune precipitate obtained from sera at the indicated time, normalized to the amount of radioactivity present in the precipitate from the pretreatment sera. 

Such antibodies were produced initially by immunization with immune precipitates, emulsified in Freund s adjuvant (2,3). Antibodies to the antigen present in the complex (and possibly to components of complement which adhere to the complex and which might exist in polymorphic forms) were formed by the recipient animal, along with antiallotypic antibodies directed to the antibody present in the precipitate. Alternatively, rabbits have been immunized with complexes of rabbit antibody with bacteria or simply with the whole IgG fraction of the donor s serum. (It is not necessary that the immunogen have known antibody activity.) The difficulty of producing an antiserum varies with the allotypic determinant. For example, it is easier to produce high titers of antibodies directed toward certain allotypic determinants on L chains than on H chains of rabbit IgG. 

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