Affinity assays

It is well known that a very important feature of many biological systems is specific recognition at the molecular level. Antibodies as a group are widely used for molecular recognition, e.g., affinity assays. This feature can be used by labeling an anti-human growth hormone antibody fraction with a fluorescent tag... 

Human liver microsome stability testing Broad-spectrum selectivity screen then, rat receptor affinity assay... 

Probes tagged with an enzyme signal generating system, such as alkaline phosphatase or horseradish peroxidase, are sometimes used in direct assays. Alternatively, enzymes as reporters may be used with biotinylated probes in a direct affinity assay or in an indirect affinity assay. For the former assay, the... 

Eaton, D.L. and Toal, B.F. (1982). Evaluation of the Cd hemoglobin affinity assay for the rapid determination of metal-lothionein in biological tissues. Toxicology and Applied Pharmacology, 66 134-142. 

Affinity assays using antibodies (immunoassays), enzymes, or nucleic acids have been established in research, clinical diagnosis, and industry for many years. However, for portable, easy to use, cost-effective devices, new sensitive receptors and measuring principles are needed. 

Although multistep affinity assays with redox-labeled targets have been described (Wang et al. [117]), most of the assays use enzyme-labeled species in conventional indirect formats (competitive, non-competitive). Direct EILAs based on multistep electrochemical affinity assays have also been developed with excellent results. In all these cases the MIP is used to extract the analyte from the sample and, after elution, the analyte is carried on to the electrochemical flow-through cell for being detected. 

Eaton DL. 1985. Effects of various trace metals on the binding of cadmium to rat hepatic metallothionein determined by the cd/hemoglobin affinity assay. Toxicol Appl Pharmacol 78 158-162. 

Affinity assays are not limited to the use of antibodies. Indeed, other kinds of molecules such as oligonucleotides or imprinted polymers can serve for the specific capture of a given marker. All these molecular moieties can be immobilised on a surface that is then placed in contact with the molecule to be detected. 

The use of ELISA is broad and it finds applications in many biological laboratories over the last 30 years many tests have been developed and vahdated in different domains such as clinical diagnostics, pharmaceutical research, industrial control or food and feed analytics for instance. Our work has been to redesign the standard ELISA test to fit in a microfluidic system with disposable electrochemical chips. Many applications are foreseen since the biochemical reagents are directly amenable from a conventional microtitre plate to our microfluidic system. For instance, in the last 5 years, we have reported previous works with this concept of microchannel ELISA for the detection of thromboembolic event marker (D-Dimer) [4], hormones (TSH) [18], or vitamin (folic acid) [24], It is expected that similar technical developments in the future may broaden the use of electroanalytical chemistry in the field of clinical tests as has been the case for glucose monitoring. This work also contributes to the novel analytical trend to reduce the volume and time consumption in analytical labs using lab-on-a-chip devices. Not only can an electrophoretic-driven system benefit from the miniaturisation but also affinity assays and in particularly immunoassays with electrochemical detection. 

The sensitivity of the detection is usually improved by the silver enhancement method. A better detection limit was reported when a silver enhancement method was employed, based on the precipitation of silver on AuNPs tags and its dissolution (in HNO3) and subsequent electrochemical potentiometric stripping detection [43]. The new silver-enhanced colloidal gold stripping detection strategy represented an attractive alternative to indirect optical affinity assays of nucleic acids and other biomolecules. 

Plasier B, Lloyd DR, Paul GC, Thomas CR, Al-Rubeai M (1999), Automatic image analysis for quantification of apoptosis in animal cell culture by annexin-V affinity assay, J. Immunol. Methods 229 81-95. 

Split-and-mix libraries have found numerous applications in the search for a substrate for a given receptor and vice versa. A typical affinity assay encompasses labeling of the host of interest with, e.g., a dye, a fluorophore, or radioactivity and equilibrating the labeled host with the bead-supported library of potential guests. The labeled host will be concentrated by those beads that carry molecules with affinity for the receptor. These beads are easily identified by visual inspection of the assay under a low-power microscope. Isolation and structural analysis reveals the structure of the active compound [3]. Recently, split-and-mix libraries are also successfully applied in the search for catalysts (see Chapter 5.4). 

The antibody capture affinity assay samples the free MAb binding sites by the addition of FITC-labeled antigen to the incubation mixture for 1 min, and the antigen-antibody complexes are immobilized on anti-IgG particles. The plates then are treated as above. 

Fig. 3. Affinity analysis of wild-type and mutant K49VlcT of MAb HyHEHO sequentially saturated with duck lysozyme in the particle affinity assay. The abscissa (R ) is the corrected proportion of antibody binding sites filled with soluble lysozyme, and the ordinate is equivalent to [Ab] times B/F, as described in the text (Reproduced from Lavoie et al.26)...

In the high affinity assay for each compound, five assay points are generated and evaluated via a Scatchard type analysis using a linearized binding equation. Rearrangement of... 

An extension of enzyme immunoassay is the enzyme affinity assay applicable to studies of nonimmunological interactions. This is already exemplified by the measurement of hormone using its receptor and by our studies on the interaction of fibronectin with collagen. - Assays of these and similar principles might well become a new area of expression for EIA. 

Ehwald R, Ballerstadt R, and Dautzenberg H. Viscosimetric affinity assays. Analytical Biochemistry 1996 234 1-8. 

To determine whether pharmacological differences are dictated by differences in nonpolar accessible surfaces of the targets within ligand-binding sites, a nonpolar distance, dnp (i,j), between the affinity-assayed kinases i, j is introduced. The dnp (i,j) is determined by differences in accessible nonpolar surface areas of the respective nonpolar hulls, Hnp ( ), Hnp (j). As rigorously defined in the previous... 

Of particular note has been the development and use of particle-based flow cytometric assays. With this technology, a flow cytometer is combined with microspheres that are used as the soHd support for conventional immunoassay, affinity assay, or DNA hybridization assay. The resultant system is very flexible and has led to the development of multiplexed assays that simultaneously measure many different analytes in a small sample volume. 

Limoges, B. Degrand, C. Ferrocenylethyl phosphate an improved substrate for the detection of alkaline phosphatase by cathodic stripping ion-exchange voltammetry. Application to the electrochemical enzyme affinity assay of avidin. Anal. Chem. 1996, 68, 4141-4148. 

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