Assay Principle

Assay principle Radioimmunoassay (RIA) Microparticle enzyme immunoassay (MEIA)... 

The assay principle for MS-based enzyme inhibition assay is shown in Fig. 5.1. The assay is based on the mass spectrometric detection of reaction products of... 

The assay principle shown in Fig. 5.10 has the potential of multiplexing, i.e. performing several assays in parallel, by pumping mixtures of receptors, i.e. streptavidin and anti-digoxigenin and reporter ligands, i.e. fluorescein-biotin and digoxin [29]. Clearly this approach will only be feasible for those assays... 

Fig. 4. Classification of reported noncompetitive immunoassays for haptens based on the assay principle. (A) Assays that include a chemical modification of hapten to allow sandwich-type detection. (B1) Improved single-antibody immunometric assays that separate immune complex and excess labeled antibody, either by using a hapten-immobilized affinity column or based on differences in their physical properties. (B2) A variation of single-antibody immunometric assays based on masking of unoccupied antibody by an immunoreactive macromolecule followed by selective capture and detection of the hapten-occupied antibody. (C) Assays employing a probe molecule specific to a hapten-antibody complex.

More recently, a somewhat similar assay principle has been reported (Gl) determining cortisol (Fig. 5B) as a model hapten, in which a polydentate ligand [multiply cortisol-substituted poly(L-lysine) with a high MW (418,400)] was used as the masking reagent (Fig. 10). First, the analyte (cortisol) and the polydentate ligand were simultaneously added to the wells on which rabbit anti-cortisol antibody had been immobilized (step i). After incubation (room... 

Self (S4) first proposed the concept of noncompetitive assay for haptens utilizing an adequate combination of an a-type and a jS-type anti-idiotype antibody, in which he used the term, selective antibody for the a-type antibodies. Then, Barnard and Cohen (Bl) applied this assay principle for the determination of serum E2, naming the assay system an idiometric assay. Figure 12A illustrates the assay procedure of the idiometric assay of E2. The target hapten is captured by excess anti-E2 antibody immobilized on microtiter strips by incubation at room temperature for 1 h (step i). After washing the strips, the /3-type anti-idiotype antibody was added in order to saturate (or block) the unoccupied paratope of the anti-E2 antibody (incubation, room temperature for 30 min) (step ii). The a-type anti-idiotype antibody, which has been labeled with a europium chelate (H4), was then added to the plate and incubated at room temperature for a further 2 h (step iii). Finally, fluorescence intensity due to bound europium was measured with a time-resolved fluorometer. Because of large steric hindrance around the bound jS-type antibody (MW 150,000), the labeled a-type antibody would. 

The assay principle should, however, be applicable to any target hapten, unlike assays based on a chemical modification. Cloning efficiency of the hybridoma-secreting anti-idiotype antibodies would be in a practical range, and much higher than that of anti-metatype antibodies. We (Kl) established four kinds of a-type and two kinds of /3-type anti-idiotype antibodies after three fusion experiments, each using spleen cells from one immunized mouse. Barnard et al. (Bl, B3, B4)... 

Very recently, a new noncompetitive assay principle called an open sandwich ELISA has been reported (S7) (Fig. 15). The assay mechanism could be regarded... 

We expect such an elegant noncompetitive assay principle, which is universally available for various types of haptens providing subfemtomol sensitivity, will be invented in the future. 

T4. Tanaka, K., Kohno, T, Hashida, S., and Ishikawa, E., Novel and sensitive noncompetitive (two-site) enzyme immunoassay for h tens with amino groups. J. Clin. Lab. Anal. 4,208—212(1990). T5. Towbin, H., Motz, J., Oroszlan, R, and Zingel, O., Sandwich immunoassay for the hapten angiotensin II. A novel assay principle based on antibodies against immune complexes. J. Immunol. Methods 181, 167-176 (1995). 

Fig. 1. Assay principle of DELFIA system using a non-luminescent I ll label.

The assay principle is straightforward the clearance of a Micrococcus hocus suspension caused by lysozyme is monitored at 450 nm and compared to a lysozyme standard of known activity. Expression of lysozyme activity in absolute terms is dependent on the substrate sensitivity. Moreover (he light scattering effect of the suspension is determined by the optical geometry of (he spectrophotometer. Therefore, absolute turbidity values can only be obtained by calibration of the apparatus with a standard of known turbidity. 

This assay principle can be applied for compounds of low and high molecular weight. 

This assay principle is advantageous in cases when labeling of small antigens renders difficult or leads to different immunological properties compared to the unlabeled antigen. 

Matrix effects lead to a decrease in binding of analyte to antibody resulting in reduced sensitivity and falsely increased or decreased concentration depending on the assay principle. The matrix effects can be reduced or eliminated by diluting the sample. 

FI is not frequently used as a readout for carboxypeptidases because the assay principle cannot be applied easily. In C terminally labeled peptide substrates, the primed site part of the car-boxypeptidase recognition sequence is missing, resulting in high KM values, incompatible with the development of robust protease activity assays. The same limitation of the FI assay principle is observed with endopeptidases in which amino acids on the primed site of the substrate (primarily PT) strongly contribute to the binding energy of the peptide. 

Strehler, Bernard L, Bioluminescence Assay Principles and Practice. 16... 

Quantitation Methods Assay Technology Assay Principle Throughput... 

In addition to the ability to work with very small amounts of reagent antibody, the indirect assay principle has the advantage that one labeled antibody may be utilized for a wide variety of assays provided only that the reagent antibody for all the different assays is raised in the same species. The materials used for the production of the labeled antibody are easy to prepare in large quantities (e.g., rabbit IgG and, say, a sheep antiserum to this) and can be carefully characterized at each stage before committing scarce materials to the assay itself. If the two-site assay procedure is used, this also avoids the use of an immunoadsorbent separation step with consequent economy in the use of the primary antigen. 

Alternatively, MIPs have also been used in biological receptors for competitive binding assays. The assay principle is similar to that in other known biological assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) except that instead of antibodies, MIPs are utilized. This method is often called molecularly imprinted assay (MIA). Typically, in MIA methods, a marker molecule (a labeled analyte analogue) is incubated together with the sample and the MIPs. Analyte and marker molecules compete for the binding... 

The assay principles of these on-site test devices are briefly summarized with some selected product listings. 

Diagnosis of the disorders discussed above requires the analysis of amino acids, organic acids, and acylcarnitines. Further details of such analytical. procedures are available on this book s accompanying Evolve site, found at http //evolve.elsevier.com/Tietz/textbook/. Here, we briefly describe the assay principles. 

Different SPA formats can be set up with these generic beads, which can be grouped into two main classes based on the assay principle (see Table 1). 

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