Immunometric assays

The emission yield from the horseradish peroxidase (HRP)-catalyzed luminol oxidations can be kicreased as much as a thousandfold upon addition of substituted phenols, eg, -iodophenol, -phenylphenol, or 6-hydroxybenzothiazole (119). Enhanced chemiluminescence, as this phenomenon is termed, has been the basis for several very sensitive immunometric assays that surpass the sensitivity of radioassay (120) techniques and has also been developed for detection of nucleic acid probes ia dot-slot. Southern, and Northern blot formats (121). 

A two-site immunometric assay of undecapeptide substance P (SP) has been developed. This assay is based on the use of two different antibodies specifically directed against the N- and C-terminal parts of the peptide (95). Affinity-purified polyclonal antibodies raised against the six amino-terminal residues of the molecule were used as capture antibodies. A monoclonal antibody directed against the carboxy terminal part of substance P (SP), covalently coupled to the enzyme acetylcholinesterase, was used as the tracer antibody. The assay is very sensitive, having a detection limit close to 3 pg/mL. The assay is fiiUy specific for SP because cross-reactivity coefficients between 0.01% were observed with other tachykinins, SP derivatives, and SP fragments. The assay can be used to measure the SP content of rat brain extracts. 

AOSD, adult onset Still disease AS, ankylosing spondylitis CA, crystal-induced arthritis ERA, enthesitis-related arthritis JA, juvenile arthritis PA, psoriatic arthritis RA, rheumatoid arthritis SE, synovium explants SLE, systemic lupus erythematosus SPCIA, solid phase 2 site chemiluminescent immunometric assay RP, relapsing polychondritis. 

In immunometric assays, unlike competitive systems, the amount of labelled antibody bound is directly proportional to the amount of unlabelled antigen present rather than inversely proportional. 

Figure 14.4. Diagram of three basic immunoassay formats, (a) Common competitive format (b) competitive assay with immobilized antigens bound to a carrier protein (immunometric assay) (c) two-site immunometric assays. Haptenic analytes are indicated as triangles, whereas larger-molecular-weight analytes are shown as teardrop shapes. Conjugated fluorescent probes are denoted by the letter "F. ...

Immunometric Assays Using an Automated How System Equipped... 

On-Line Immunometric Assays Based on Nonimmunological Removal... 

Immunometric Assays Based on Masking Unoccupied Antibody Binding Sites. . 155... 

Fig. 3. Schematic representation of noncompetitive immunoassays. (A) Two-site immunometric assays (sandwich assays) and (B) single-antibody (single-epitope) immunometric 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.

Several noncompetitive assays for haptens reported so far can be regarded as variations of conventional single-antibody immunometric assays, the majority of which (the assays in Sections 3.1 and 3.2) are based on principle B1 in Fig. 4. In many cases, these methods employ an automated flow system to simplify the assay procedure and minimize the time and labor required for the analysis. 

The modified single-antibody immunometric assays discussed below are based on principle B2 in Fig. 4. In these assays, unoccupied antibody-binding sites are masked by reaction with a multiply hapten-labeled macromolecule to permit subsequent selective determination of hapten-occupied antibodies. 

This immunometric assay employed an analog of the analyte (T2) as a ligand of immunosorbent, as in the example discussed in Section 3.1. The authors mentioned that the use of T2 molecules having low intrinsic affinity to the antibody but high avidity in the immobilized form on the CPG, through the use of divalent binding, obviated the need to prepare a monovalent labeled antibody. 

A prominent advantage of this assay procedure is the feature that the complex of hapten and labeled antibody was captured on a solid phase (PMP) and separated from the reaction medium before signal determination. This additional step not only reduces interference due to biological specimens but also eliminates the tedious transfer of supernatant, which is essential in conventional immunometric assays. This immunometric assay provided somewhat improved specificity in terms of the cross-reactivities with T2 and reverse T3 (3,3, 5 -L-triiodothyronine). The authors speculated that the dissociation rate of the antibody-cross-reactant complex would be faster than that of an antibody-analyte complex thus the former binding would be preferentially substimted by T2 immobilized on CPG. 

Using an antibody specifically recognizing the antigen-antibody complex, more direct noncompetitive hapten immunoassays, which can be regarded as semi two-site immunometric assay, could be established (S3). Figure 14 depicts two typical procedures of noncompetitive assays using anti-metatype antibodies, which are based on principle C in Fig. 4. 

B6. Buscarlet, L., Grass , J., Creminon, C., Pradelles, R, Dupret-Carruel, J., Jolivet, M., and Mons, S., Cross-linking of 17/i-estradiol to monoclonal antibodies by direct UV irradiation Application to an enzyme immunometric assay. Anal. Chem. 71, 1002-1008 (1999). 

E2. Freytag, J. W., Dickinson, J. C., and Tseng, S. Y, A highly sensitive affinity-column-mediated immunometric assay, as exemphfied by digoxin. Clin. Chem. 30, 417-420 (1984). 

G2. Grassi, J., Creminon, C., Frobert, Y., Etienne, E., Ezan, E., Volland, H., and Pradelles, R, Two different approaches for developing immunometric assays of h tens. Clin. Chem. 42,1532—1536... 

In non-competitive or reagent excess immunoassays [26], an excess of immu-noreagent (antibody or antigen) is used so that all the analyte forms an immunocom-plex that is further quantified and related to the analyte concentration in the sample. These assays are also known as immunometric assays and their advantages over competitive assays include higher sensitivity, precision, and analyte working range. 

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