Immunochemical precipitation

Immunochemical assay procedures may be divided into two classes, direct and indirect. Direct methods involve synthesis or modification of the molecule to be measured in the presence of a radioactively labeled precursor followed by its isolation using immunochemical precipitation. Indirect methods involve competition of a nonradioactive protein obtained from an experimental sample with a radioactively labeled standard protein for a limited amount of antibody the quantity of desired protein... 

Nearly all current methods of isolation and purification of lectins rely on affinity chromatography. Naturally, the characteristic ligand must be determined in advance. The properties of lectins can be used to precipitate macromolecules and to agglutinate some types of cells, be they plant or animal. The driving force of this reaction is the association with certain bound residues, generally monosaccharides, from the macromolecule or the cellular periphery. When this type of reaction is observed, the problem is to find the sugar that can inhibit activity at the lowest possible molar concentration. As in the case of immunochemical precipitations, this inhibition is due to the occupation of the recognition site by the small soluble molecule. 

A polysaccharide obtained from certain seaweeds which forms a gel when water is added. It is composed of at least two fractions, agaropectin and agarose. Agar gels are used in many immunochemical precipitation reactions and can also be used as an electrophoretic medium, although agarose is preferred for this particular purpose. 

Oudin, J. (1980) Immunochemical analysis by antigen-antibody precipitation in gels. Methods Enzymol. 70,166-198. 

Immunoprecipitation (IP) is one of the most widely used immunochemical techniques. It involves the interaction between an antigen and its specific antibody. Antigen-antibody interactions may produce a network of many antigen molecules cross-linked by antibody molecules, which result in insolubilization and precipitation of the complex (Williams 2000). 

Addition of appropriate amounts of salts, such as ammonium or sodium sulfate (2), or other chemicals, such as PEG, cause precipitation of IgG from serum. Caprylic acid can also be used to fractionate proteins from serum Although such IgG is usually contaminated with other proteins, the ease of these precipitation procedures coupled with the high yield of IgG produced has led to them being very widely used to produce enriched IgG preparations suitable for many immunochemical procedures, e.g., production of immunoaffinity columns, and as a starting point for further purification. The precipitated IgG is usually very stable and such preparations are ideally suited for long-term storage or distribution and exchange between laboratories. 

Antibody fragment preparations or PEG-precipitated phage can be used as immunochemical reagents. 

Figure 2. Immunochemical studies ofthe specificity ofrabbit antibodies elicited against Parp(al—3)D-Manp-BSA glycoconjugates (02). Top structures of the Salmonella O-antigen-specific saccharides used. Center Inhibition of precipitation in mixtures of anti-PM-BSA serum andS. paratyphi A PS. Key Center V, Octasaccharide S. paratyphi A. var. Durazzo ft.. Dodecasaccharide S. paratyphi A. var. Durazzo (II) , Parp D-Manp 4 OMe a., Parp -jpi- D-Manp O-0-NHCSNH(CH2)5COOH (VIII) m. Parp -j-i- D-Manp O-0-NHCOCF. (VII) U, Parp OMe A, Dodecasaccharide...

The development of immunoassays for the detection of food components and contaminants has progressed rapidly in the last few years [7]. Antibodies against almost all the important food residues compounds are currently available. Classical immunochemical methods such as immunodiffusion and agglutination methods for food analyses generally involve no labeled antigen or antibody. Concentration of the antigen-antibody complex is estimated from the secondary reaction that leads to precipitation or agglutination. These methods are not sensitive, are subject to... 

Examination of cryoglobulins especially in patients with monoclonal IgM requires collection of at least 10 ml of blood into a prewarmed (to 37°C) tube or syringe, allowing the blood to clot at that temperature for 30-60 min prior to centrifugation to obtain serum. The serum sample should be stored at 4°C for up to 7 days. The presence of cryoglobulins in a formed precipitate should be confirmed by its solubility at 37°C, and further quantitated and characterized immunochem-ically (Kl). 

The immunochemical studies of Kabat and coworkers200 686 revealed some striking subtleties in Lotus tetragonolobus sugar-specificity. Precipitin reactions between the lectin and several blood-group H substances demonstrated strong affinity of the lectin for this determinant. H substances precipitated 83-88% of the purified Lotus lectin at... 

Figure 11.8. Detection of NOS-dependent S-nitrosylation in vivo, a Immunochemical detection procedure. A primary antibody directed against nitrosothiol groups was used in combination with an enzyme-linked secondary antibody. The dye released by the enzyme is insoluble and thus will precipitate close to the site of formation, b S-nitrosylationinaorticrings. Samplesweie treated with Phenylephrine (PE), acetylcholine (Ach), and the inhibitor N-methylarginine (L-NAME) as indicated. Brown stain deposits indicate S-nitrosylation. The traces above the histology panels illustrate the contraction and relaxation responses evoked by the dmgs.

Most, if not all, contemporary applications of immunochemical techniques are based on the reaction of antibodies with the antigen used to induce their production, to yield a very stable complex. If both antigen and antibody are present in solution, a precipitate forms as long as the antibody is in molar excess. This is known as a precipitin reaction and may be quantitated as shown in Figure 8-15. For this example a highly purified preparation of avidin was injected into a rabbit which then produced avidin-specific antibodies. Since biotin forms a very stable complex (dissociation constant with avidin, complexation of... 

Identification of the individual precipitates in a pattern may be carried out immunochemically since a large number of monospecific andsera are now commercially available and can easily be used for idendficadon purposes seelor example. Note 9 on CIE with a trap-gel ). The tandem CIE also detailed in Note 10 can be another way to idendfy a precipitate if pure known andgen is available. 

The predominant contribution of the folded native structure is amply illustrated by the findings that complete reduction of disulfide bonds and 5-carboxymethylation completely alters the immunochemical reactivity of protein antigens, such as ribonuclease, lysozyme, and other antigens.However, reduction of but two of the four disulfide bonds in ribonuclease had a negligible effect on the ability to precipitate with antibody to native ribonuclease. 

Immunochemical Analysis by Antigen-Antibody Precipitation in Gels... 

The author s aims are a) to classify as rationally as possible the techniques of immunochemical analysis by antigen-antibody precipitation in gels (,b) to state the more or less general principles and laws which hold true for all or part of the techniques (c) to help the reader in understanding the bases of the different techniques in a way that does not require any special knowledge of physics and (d) to give a few examples to illustrate that this understanding of the principles and laws, plus an analysis of the reactions may raise, and eventually solve, problems of appreciable importance. 

Approximately 2 years after antigen-antibody precipitation in gels was first proposed for immunochemical analysis, a number of techniques began to appear in the literature. No attempt is made to describe or enumerate all these techniques or their variations, which are described elsewhere. ... 

In contrast to the techniques described, several procedures use antigen-antibody precipitation in gels as a means of immunochemical analysis with an electric field as the motor of the reagents. 

Kla. Kabat, E., Bendich, A., and Bezer, A. E., Immunochemical studies on blood groups. II. Properties of the blood group A substance from pools of hog stomach of specific precipitates composed of A substance and homologous human antihody. J. Exptl. Med. 83, 447 (1946). 

Non immunochemical Turbidimetric and Nephelometric Methods. Precipitation of protein for turbidimetric or nephelometric assays is achieved with sulfosahcylic acid alone, or with sulfosahcyfic acid in combination with sodium sulfate or trichloroacetic acid (TCA), or with TCA alone. Precipitation methods for total protein assay depend on formation of a fine precipitate of uniform, insoluble protein particles, which scatter incident light in suspension. 

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