Conjugation, reagents

Figure 17.22 Certain unique phosphine derivatives can be used in the design of modification or conjugation reagents to create a traceless Staudinger ligation process, wherein the phosphine group is lost and an amide bond between an azide-containing molecule and the phosphine-containing molecule results.

Figure 19.1 Immunogens are made by the crosslinking of a hapten molecule with a carrier using a conjugation reagent.

The RPIA technology has been enhanced in the Stratus CS system by utilization of a dendrimer-antibody complex in which the analyte-specific capture antibody is covalenty coupled onto a dendrimer. The test packs in the Stratus CS system include dendrimer-capture antibody complex reagent, the alkaline phosphatase labeled antibody conjugate reagent, the substrate-wash reagent and a piece of glass fiber filter paper as the solid phase. Preparation and unique properties associated with these dendrimer-coupled antibody complexes are described below. 

In addition, great care should be taken with the storage and the use of the antibodies and conjugating reagents. Freeze-thaw cycles should be kept to a minimum by either aliquoting antibody stock solutions into working aliquots or into aliquots which can be kept at 4°C. It is also always a good idea to keep the antibody aliquots on ice while in use... 

Receptor-specific antibodies and fluorochrome-conjugated reagents are diluted in PBS-gelatin or PBS-gelatin-Sap (for permeabilized cells) as required. Appropriate antibody dilutions should be determined empirically. Note Cells should not be allowed to dry at any stage in the procedure. 

Conjugation reagents Formamide (>99.5%, Fluka) BisTris HBr, ammonium acetate. HPLC columns are as for peptide and PNA syntheses and columns are immersed in a water bath (45°C) or surrounded by avery efficient heating jacket. 

Some important methods of protein labeling utilize various conjugation reagents 130-136) jjjg common advantage of these methods is that they are relatively... 

Halogenation has been already described (see 2.1). In conjugation methods a radionuclide is included in the molecule of a conjugation reagent which then reacts with the protein to be labeled. The label introduced into the protein molecules... 

Conjugation reagents bind to protein functional groups NHj-, SH-, OH-, phenol, etc. Reagents which react with the amino groups are usually preferred. The utilization of SH-groups could be risky because they can be included in the catalytic site of an enzyme or participate in some other way in the biological activity of protein to be labeled protein cannot occur. The presence of a radiolabel outside of the peptide active site. 

Some reagents bind to several functional groups of proteins, e.g. dansylchloride, acetic anhydride. The list of the most frequently used conjugation reagents is given in Table 7. The most important ones are briefly characterized below. 

First prepared by Rudinger and Ruegg the Bolton-Hunter reagent is one of the most frequently used conjugation reagents. Its iodinated form has been prepared by Bolton and Hunter . Chemically it is N-succinimidyl-3 (4-hydroxy,5- I iodo-... 

Table 7. Survey of the most frequent conjugation reagents used for protein labeling...

Fig. 3. Formulae of most common conjugation reagents f I]-Iodosulfanilic acid (left) Methyl 3,5,-di-[ I]-iodohydroxybenzimidate (middle)...

Table 2 Conjugating Reagents Used for Various Functional Groups...

Conjugate reagent The anti-IBDV monoclonal antibody (mAb) is purified by a protein A column from ascitic fluids of mice carrying the specific hybridoma (5), and stored at -20°C in lmL aliquots. 

Conjugate reagent The excretory-secretory (ES) antigens are purified from the supernatants of cultured Trichinella muscle larvae (7), and stored at -70°C in 1 mL aliquots. 

The following synthetic route gives access to disilyl conjugative reagents, either as the acetate or the methyl carbonate. 

The use of immunoassay techniques for the determination of PAHs has been reviewed. Immunoassay is based on the coupling of a specific biological antibody in the detection device with the analyte either directly in water or extracted from solid samples and diluted in buffer solution. Enzyme-linked immunosorbent assay (ELISA) is the most common immunoassay technique employed in commercially available test kits. Water samples or soil extracts are added with an enzyme conjugate reagent to immobilized antibodies where the conjugate competes with PAHs for binding to the antibodies. ELISA test kit sensitivity and crossreactivity depends on the PAH used to raise the antibody. Antiphenanthrene or antffluoranthene antibodies raised in host animals are the most commonly employed. Test kits will be most sensitive to the PAH from which the antibody was... 

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