Assay formats excess reagent

The double-antibody sandwich technique is applicable to large molecules. Small analytes have difficulty forming the double-antibody sandwich immunocomplex. The smallest analytes reported that have been used in a double sandwich assay are peptides of around 10 amino acid residues (158). The double-antibody sandwich technique measures the occupied antibodies using an excess-reagent assay protocol. A limited-reagent assay protocol can also be designed, as shown in Fig. 4. In this format, the antibodies are immobilized onto the solid phase,... 

The only rate-limiting factor in a coupled assay should be the concentration of the initial and linking products and all other reagents should be in excess. The role of the auxiliary and indicator enzymes is essentially that of a substrate assay system and under optimum assay conditions the rate of the indicator reaction should be equal to the rate of formation of the initial product. The indicator reaction must be capable of matching the different test reaction rates and its velocity can be defined by the Michaelis-Menten equation in the usual way ... 

An indirect vanadometric method of assay for camphor was developed. The method involves the formation of 2, -dinitrophenylhydrazone of camphor. The nitro groups in the hydrazone are then reduced to amines by treatment with vanadium sulfate (VSOl ) and the excess of the reagent is back titrated with sodium dichromate (NapCrpO-y). The entire procedure is carried out in a modified separatory funnel into which standard vanadate, 6tJ and zinc amalgam... 

Noncompetitive Format. Noncompetitive assay is based on migration differentiation between the Ag-Ab complex and unbound reagent(s) provided by CE. An excess amount of a reagent is added to the sample, which contains a certain amount of an analyte. The affinity reaction which occurs in this case can be described by the well-known equations... 

Certain aromatic amines may cause reduction of Fe(III) ions in the presence of Fer-roZine Iron reagent (125), according to equation 18, leading to the formation of a stable violet-colored complex with Fe(II) ions (126), the concentration of which is measured at 562 nm. At pH 5, the color develops in a few minutes at room temperature and the absorbance of 126 is proportional to the concentration of the analyte. Neither the oxidation products of the analyte nor the excess of Fe(III) or 125 affect the assay however, not all aromatic amines undergo this reaction. Thus, compounds such as 1,4-phenylenediamine, 2,4-diaminotoluene, 8-aminoquinoline and 2-amino-3-hydroxypyridine can be determined... 

To detect the assay product, it is usually necessary to use a label, which is attached either to the antibody or the antigen. This label can be fluorescent, luminescent, radioactive, an enzyme or an electrochemically active group. Immunoassay reactions can be performed in a large variety of formats, in solution or on a solid support, with limited reagent or an excess of reagent. These formats are discussed in more detail in the following sections, after a description of antibody and antigen structure and immunocomplex formation. 

The substrate is cleaved by the enzyme-labeled hapten in the immunocomplex at the surface. The fluorescence can be measured in solution or at the surface. For the determination of high molecular mass antigens a two-site sandwich assay can be applied. A variation represents the double antibody assay, whereby the second antibody, which is directed against the hapten-specific antibody, is enzyme-labeled. Reagent-excess based assays are mainly used for the determination of antibodies in a noncompetitive format. In Scheme 6, a procedure for an indirect ELISA for the determination of antibodies is described. 

The tetraphenylboron method for small quantities of bases, given under Atropine, p. 116, has also been applied successfully to some preparations of local anaesthetics. Procaine in Injection of Procaine and Adrenaline can be determined by diluting 2 ml of sample to 20 ml with dilute buffer solution, pH 3 7, and taking 10 ml for the assay each ml of cetylpyridinium chloride is equivalent to 0 001364 g. Amethocaine, however, tends to give high results if the reagent is added in large excess, probably due to the tendency to partial formation of a di-tetraphenylborate. 

Oxidation is conducted in an acidic medium, usually an acetate or acetate-formate buffer, by using a controlled amount of NBS. Side reactions usually encountered with NBS and proteins, such as oxidation of methionine, cysteine, cystine, histidine and tyrosine, have been generally assumed to be minimal and not to interfere with the tryptophan determination. Experiments with model compounds confirm that the reactivity toward NBS of tryptophan is much higher than that of other amino acid residues. Oxidation of residues other than tryptophan is revealed by increased consumption of NBS by the protein. Usually, 2-4 equivalents of reagent are needed for complete oxidation of tryptophan, but with some proteins, an excess of up to 20 equivalents may be needed (230, 372,374). Some proteins possess tryptophan residues which react very slowly or not at all under the usual conditions of assay the reaction should then be carried out in 8 M urea. 

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