Tracers haptens

Table 2 Guidelines for design of coating/tracer haptens...

To a solution of known titre antiserum is added a known concentration of radioactive or tracer hapten (antigen) and an aliquot of the plant extract. There will be a competition of the labelled hapten (added) and unlabelled (plant extract) hapten for the fixed number of antibody sites (antiserum of known titre) and this results in some of the labelled hapten being bound while the remainder remains free. The distribution of the radioactive hapten between the free and bound state will be a function of the amount of unlabelled hapten present in the assay tube. 

Figure 14.3. Distinction between homogeneous and heterogeneous immunoassay formats. Haptenic analytes are indicated as triangles, and conjugated fluorescent probes are indicated by the letter F. In this hypothetical depiction, the homogeneous immunoassay is quantitated in the original reaction mixture. The heterogeneous immunoassay requires removal of unreacted tracer, further addition of reagents such as an enzyme to release a fluorescent molecule F, followed by quantitation.

It is not necessary that the tracer or radioligand is structurally identical to the pesticide of interest. The same considerations used in deciding where to attach a hapten to a protein should be applied to attaching a hapten to either a commercially available labeled compound or to a compound which is easily labeled in a subsequent step. For instance, the conjugation of the hemisuccinate of S-bioallethrin to commercially available 3H tyramine (j>-[2-aminoethyl]phenol) led to a useful radioligand (6, 7). ... 

Success in the synthesis of new catalytic antibodies (CAs) depends on the efficiency of each of the following steps 1) hapten design, 2) immunogen synthesis, 3) preparation of the enzymatic tracer 4) generation and purification of antibodies and 5) kinetic assays. 

The easiest way is to introduce a radioactive tracer. The number of residues attached to each molecule of the carrier can then be determined by comparing the specific activity of the hapten and the conjugate. ... 

Antigens, haptens, and antibodies radiolabeled with or are commonly used as tracers in immunoassay. These nuclides can be introduced directly into functional groups normally present in proteins and other macromolecules or into suitable derivatives that can be synthesized by a variety of chemical procedures. The most widely used iodination methods have been direct chemical or enzymic substitution of hydrogen in tyrosine or related groups using chloramine-T or lactoperoxidase, respectively. These methods are described in separate chapters in this volume. 

I-labeled protein A as a tracer for IgG has been extended to a general immunoassay method for fluid-phase antigen and hapten. The steps involved are summarized in Eqs. (2) and (3). 

I-labeled protein A can be used as a tracer for IgG antibodies in RlAs for antigens and haptens. See this volume [25]... 

The model considers that the equilibrium constants for the analsrte and competitor are of the same order of magnitude, which often is not the case for small hapten analsrtes due to difference in structure of tracer and analyte. 

Hapten molecules are often coupled to carrier proteins (e.g., coating antigens used for the development of indirect competitive assays) or to enzjune labels (e.g., tracers used for detection in competitive assays). A protein has usually many different sites to which the hapten can be coupled and the chosen chemistry should obviously be one that leaves the active site as httle affected as possible. The number of haptens that can be coupled to one protein molecule (hapten density) by a particular chemistiy is essential for the final assay outcome [106-110]. 

Restricted-access-based materials (RAM) can trap small hapten-based tracers, where the fraction separation is governed both by size exclusion and hydrophobic interactions [60,62,86,122,123]. There are indications that the RAM phase competes with the antibody-bound tracer and analyte [124], as shown in Fig. 9.18. 

Conjugate Antigen/antigen derivative or antibody covalently coupled to a label (= tracer) or hapten coupled to a carrier protein molecule (= immunogen)... 

Steroid chemistry is involved at two points (i) the preparation of a steroid derivative ( hapten e.g. a carboxymethyloxime or a hydrogen succinate ester) suitable for chemical combination with free amino-groups in BSA (see below) and (ii) the synthesis of labelled steroids of high specific radioactivity for use as tracers. 

Several ELISA formats have been considered, and the one currently being tested is called the "hapten tracer" method. First introduced to this project by Dr. Freya Jung while a postdoctoral fellow in Dr. Hammock s laboratory, this method uses microtiter plates coated with a commercial preparation of goat anti-mouse antibody. Enzyme-labeled hapten competes with analyte for receptor sites on a mouse monoclonal antibody specific for the analyte. The amount of enzyme left after washing is inversely proportional to the... 

Antibodies with high specificity for 4-hydroxy-oestrogens have been obtained by use of a 17-(0-carboxymethyl)oxime-BS A conjugate as antigen the same hapten combined with [125I]iodohistamine was employed as radioactive tracer.135 Antisera... 

Haptens and anti-hapten antibodies are reagents which can be used in a wide variety of assay formats. One fundamental division among immunoassay formats is based on the type of label or tracer which is ultimately detected. Two of the most common types of label are radioisotopes and enzymes, used respectively in radioimmunoassay (RIA) and enzyme immunoassay (EIA). RIA is the older of the two and remains very important and widely used in clinical and research situations. However, the use of radioisotopes carries with it possible health hazards, the need for special handling precautions, and mandatory regulatory oversight. Because EIA avoids these problems, it is gaining popularity rapidly among users of immunoassays. 

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