Noncompetitive immunoassay

Following principles similar to those of immunoassays, noncompetitive hybridization assays in general demonstrate better sensitivity than competitive formats of... 

Yang WC, Schmerr MJ, Jackma R et al (2005) Capillary electrophoresis-based noncompetitive immunoassay for the prion protein using fluorescein-labeled protein A as a fluorescent probe. Anal Chem 77 4489 1494... 

The reagent also has been used in a unique tRNA-mediated method of labeling proteins with biotin for nonradioactive detection of cell-free translation products (Kurzchalia et al., 1988), in creating one- and two-step noncompetitive avidin-biotin immunoassays (Vilja, 1991), for immobilizing streptavidin onto solid surfaces using biotinylated carriers with subsequent use in a protein avidin-biotin capture system (Suter and Butler, 1986), and for the detection of DNA on nitrocellulose blots (Leary et al., 1983). 

Q Wang, G Luon, J Ou, WSB Yeung. Noncompetitive immunoassays using protein G affinity capillary chromatography and capillary electrophoresis with laser-induced fluorescence detection. J Chromatogr A 848 139-148, 1999. 

CAE employing antibodies or antibody-related substances is currently referred to as immunoaf-hnity capillary electrophoresis (lACE), and is emerging as a powerful tool for the identification and characterization of biomolecules found in low abundance in complex matrices that can be used as biomarkers, which are essential for pharmaceutical and clinical research [166]. Besides the heterogeneous mode utilizing immobilized antibodies as described above, lACE can be performed in homogeneous format where both the analyte and the antibody are in a liquid phase. Two different approaches are available competitive and noncompetitive immunoassay. The noncompetitive immunoassay is performed by incubating the sample with a known excess of a labeled antibody prior to the separation by CE. The labeled antibodies that are bound to the analyte (the immuno-complex) are then separated from the nonbound labeled antibody on the basis of their different electrophoretic mobility. The quantification of the analyte is then performed on the basis of the peak area of the nonbonded antibody. 

NONCOMPETITIVE IMMUNOASSAYS FOR SMALL MOLECULES WITH HIGH SENSITIVITY AND SPECIFICITY... 

Noncompetitive Hapten Immunoassays Employing Chemical Derivatization... 

Noncompetitive Hapten Immunoassays Using New-Generation Antibody Reagents... 158... 

Fig. 2. Typical dose-response curves of (A) competitive immunoassays and (B) noncompetitive immunoassays.

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

Noncompetitive Hapten Immunoassays Employing Chemical Derivatization ot an Analyte Permitting Sandwich-Type Determination... 

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.

Fig. 6. Principle of a noncompetitive hapten immunoassay based on quantitative biotinylation of the target molecule (A) biotinylation of a hapten with reactive amino group(s) and subsequent (B) sandwich-type immunoassay procedure.

Fig. 9. Schematic representation of the noncompetitive immunoassay of triiodothyronine using a solid-phase immobilized hapten for masking an unoccupied antibody.

The invention of B cell hybridoma technology (K3) has allowed the generation of various kinds of useful antibodies, even very minor or rare antibody species elicited in serum by the conventional procedure, as a pure immunochemical reagent in almost unlimited amounts. Among such new-generation monoclonal antibodies, anti-idiotype antibodies and anti-immune complex (anti-metatype) antibodies have been successfully introduced as key reagents enabling noncompetitive hapten immunoassays (Fig. 11). 

Fig. 12. Noncompetitive hapten immunoassay procedures (A and B) using a combination of the a-type and j6-type anti-idiotype antibodies, each recognizing the framework and paratope of the anti-hapten antibody. Anti-hap, anti-hapten antibody (primary antibody) a-Id, a-type anti-idiotype antibody /J-Id, /i-type anti-idiotype antibody S, signal-generating group B, biotin SA, streptavidin.

We (K1) attempted to develop a noncompetitive assay based on the anti-idiotype antibodies for a conjugated bile acid metabolite, ursodeoxycholic acid 7-A-acetyl-glucosaminide (UDCA 7-NAG), which is expected to serve as a diagnostic index for an autoimmune disease, primary biliary cirrhosis. In our assay, the hapten UDCA 7-NAG, a /3-type antibody, and a biotin-labeled a-type antibody were simultaneously added to a microtiter plate coated with an F(ab )2 fragment of a specific anti-UDCA 7-NAG antibody, then incubated at room temperature for 8 h. Bound biotin was then detected with HRP-labeled streptavidin, whose enzyme activity was measured using o-phenylenediamine/H202 as a substrate. This noncompetitive assay system provided a subfemtomole-order sensitivity (detection limit 118 amol) that was 7 times lower than the competitive immunoassay using the same anti-hapten antibody (K2), even with a common colorimetric detection (Fig. 13). Somewhat improved specificity was also obtained namely, better... 

Dose-response curves of (O) noncompetitive and ( ) competitive immunoassays of UDCA... 

Cross-Reaction of Noncompetitive and Competitive Immunoassays for UDCA 7-NAG... 

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. 

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