Immunoassay noncompetitive assays

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... 

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. 

Noncompetitive immunoassays This class comprises the following subtypes excess reagent, two-site and sandwich assays. A typical noncompetitive assay has the following major components (i) primary or capture antibody, (ii) sample... 

Noncompetitive Immunoassays. In a typical noncompetitive assay for an antigen, a capture antibody is first passively adsorbed or covalently bound to the surface of a solid phase. Various sequences in which the capture antibody can be attached are shown in Box 9-2. The simplest involves direct attachment to the solid phase. However, this can lead to some loss of antibody binding capacity because of steric factors or attachment of the antibody via its Fab region. To protect the binding properties of the antibody, more complex sequences have been devised. For example, the solid support can be coated with an antispecies antibody, and then... 

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

Immunoassays are classified into two major groups, depending on whether assay reagents are used in excess (noncompetitive assay, Figure 12.2) or are a limiting factor (competitive assays, Figure 12.3). 

Methods very similar to classical immunoassays in the sandwich format are easily implemented in flow systems (Fig. 2d). In this type of noncompetitive assays, again, antigen is captured and concentrated from an appropriate volume of sample on an immunosorbent (-Abi) column while nonantigenic components are eluted. Subsequent to the capture step, labeled second antibody (Abj-label) is introduced into the mobile phase and swept into the column, where it binds to the -Ab]-Ag complex to form -Ab -Ag-Ab2-label. Unbound Ab2-label is swept from the column, and when the label is an enzyme, antigen is quantitated indirectly by conducting an enzyme assay in the column. After substrate incubation, the reaction product is transported to a detector at the column terminus. Ag and Ab2-label can be introduced in the column sequentially or simultaneously. In some instances both modes led to similar sensitivity [55], and in other cases simultaneous injection produced a greater response than sequential injection [56]. The term sandwich has also been applied to the procedure carried out to quantitate Ab by capturing a complex Ab-Ag-label onto a protein G capillary column [57]. In this case detection is performed after elution. 

There are three main formats of immunoassays direct noncompetitive assays, competitive (direct or indirect) assays. 

One popular method of immunoassay is a competition assay coupled with an optical or an electrochemical transducer. Similar to direct noncompetitive assay, either antibody (Figure 30b) or antigen (Figure 30c) can be... 

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.

Bl. Barnard, G., and Kohen, R, Idiometric assay Noncompetitive immunoassay for small molecules typified by the measurement of estradiol in serum. Clin. Chem. 36, 1945-1950 (1990). 

HI. Hara, T, Nakamura, K., Satomura, S., and Matsuura, S., Noncompetitive immunoassay of thyroxine using a liquid-phase binding assay. Anal. Chem. 66, 351—354 (1994). 

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). 

Immunoassays, electrochemical — A quantitative or qualitative assay based on the highly selective antibody-antigen binding and electrochemical detection. Poten-tiometric, capacitive, and voltammetric methods are used to detect the immunoreaction, either directly without a label or indirectly with a label compound. The majority of electrochemical immunoassays are based on -> voltammetry (-> amperometry) and detection of redox-active or enzyme labels of one of the immunochemical reaction partners. The assay formats are competitive and noncompetitive (see also -> ELISA). 

Both competitive and noncompetitive methods have been incorporated into homogeneous enzyme-labeled immunoassay kits that ultimately relate enzyme activity to analyte concentration.22 The competitive-binding assays are called enzyme-multiplied immunoassay technique (EMIT), substrate-labeled fluorescein immunoassay (SLFIA), apoenzyme reactivation immunoassay (ARIS), and cloned enzyme donor immunoassay (CEDIA), while a noncompetitive method is called enzyme inhibitory homogeneous immunoassay (EIHIA). 

The analytical detection limits of competitive and noncompetitive immunoassays are determined principally by the affinity of the antibody and the detection limit of the label used, respectively. Calculations have indicated that a lower limit of detection of lOfmol/L (Le., 600,000 molecules of analyte in a typical sample volume of 100 jiL) is possible in a competitive assay using an antibody with an affinity of iO L/mol. Table 9-2 illustrates the detection limits for isotopic and nonisotopic labels. A radioactive label, such as l, has low specific activity (7.5 million labels necessary for detection of 1 disintegration/s) compared with enzyme labels and chemiluminescent and fluorescent labels. Enzyme labels provide an amplification (each enzyme label produces many detectable product molecules), and the detection limit for an enzyme can be improved by replacing the conventional photometric detection reaction by a chemiluminescent or bioluminescent reaction. The combination of amplification and an ultrasensitive detection reaction makes noncompetitive chemiluminescent EIAs among the most sensitive types of immunoassay. Fluorescent labels also have... 

More sensitive and noncompetitive immunoassays (IRMAs) have also been developed. Currently available assays use antibodies against PTHrP (sequences 37-74,1-40, i-40, 1-34, 1-40, and so on) as capture antibodies. Their radiolabeled signal antibodies are against PTHrP (sequences 1-36, 60-72, 57-80, 37-67, 50-83, and so on), respectively. The limit of detection for these assays is reported to be from 0,1 to 1.0 pmol/L. 

Immunoassays can be designed in two formats competitive assays, preferable for quantification of small molecules such as steroid hormones and prostaglandins, and noncompetitive, or sandwich, assays restricted almost exclusively to large molecules... 

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