Labels in immunoassay

In the original immunoassay of Yalow and Berson (5), the label of choice was a radioisotope and today radioisotopes are still widely used as labels (see The Immunoassay Index I, Lab. Pract. 32(8) (1983) 21), I being the most commonly used. Radioimmunoassay (RIA) has been used extensively in the determination of steroid and peptide hormones, drugs (both for therapeutic monitoring and for the detection of drugs of abuse) and various macromolecules of clinical interest. 

The principal advantages of radioimmunoassay are the selectivity of the assay, conferred as in all immunoassays by the specificity of immunological reactions, and the sensitivity of radioisotope determination. The latter is the factor that has led to RIA becoming the most extensively used form of immunoassay, as background interference is not a problem. 

However, RIA does have several major disadvantages which have led to extensive research into the use of alternative labels. The most important of these include (i) the inherent instability of radiolabelled compounds (ii) the need for specialized equipment and associated safety precautions (iii) the cost of preparing radiolabelled compounds (iv) the heterogeneity of all RIAs, because the properties of the label do not change on Ag-Ab binding, hence automation on a small scale is difficult (v) disruption of the reaction by radioactivity. 

When chosing a non-isotopic label for immunoassay perhaps the most important factor to be considered is the level at which it can be determined in the matrix of analytical interest. Other factor include (i) freedom from hazards (ii) ease of introduction into the sample molecule (iii) cost and stability (iv) suitability for homogeneous assays and automation (v) possibility of combination with another label to develop a multicomponent analysis (vi) application of the method in sensor development. 

Alternative labels that have been investigated are reviewed in Table 6.1. As can be seen from the table, certain of these methods along with RIA are unsuitable for application to sensor development and will not be mentioned further. Other methods, particularly fluorescence immunoassay (FIA), will be discussed in more detail later with particular reference to their application in sensors. 

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One of the spinoffs of this work has been the appreciation that bio- or chemiluminescence could be used as an aid in analytical chemistry. This has led to the successful applications of acridinum esters as labels in immunoassays [6]... 

As indicator enzymes horseradish peroxidase (HRP or HRPO), alkaline phosphatase (AP), or /i-galactosidase, are favored, since they are relatively robust, have a high product-forming rate, are easy to purify, and are cheap. The most used colloids are from gold, silver, and iron, and iodine isotopes are mostly taken as radioactive labels in immunoassays. 

Nanoparticles, e.g., silicon, gold, silver (Dequaire et al., 2000), are often used as materials for protein labeling in immunoassays, especially in lateral flow device (LFD), where they are responsible for visualization of the results. Their advantage lies in the fact that they enhance the optical signal, and reduce the background interference (Schneider et al., 2000 Lochner et al., 2003 Matveeva et al., 2005 Chumbimuni-Torres et al., 2006 Li et al., 1999 Peng et al., 2007a). 

There is increasing interest in the use of nonisotopic labels in immunoassay. The main advantages of such labels in labeled antibody techniques are the expected longer shelf life of the reagent and the ability to use a detection apparatus that may for one reason or another prove to be more convenient—e.g., for automation or cheapness. Some alternatives that are being explored are bacteriophages, spin labels, enzymes, fluorescent compounds, and luminescent compounds. ... 

CALCIUM-REGULATED PHOTOPROTEIN OBELIN AS A LABEL IN IMMUNOASSAY AN OUTLOOK FOR APPLICATIONS... 

So there were obvious implications to apply obelin as a label in immunoassay the protein accessibility, high sensitivity, an unlimited linear range of bioluminescence, the lack of background, the simplicity with which the reaction is triggered, the absence of hazard and the availability of modem registration devices. 

Calcium-Regulated Photoprotein Obelin as a Label in Immunoassay... 

Using the genetic approach the biotinylated obelin was obtained. A recombinant apoobelin capable of being biotinylated in vivo in E. coli cells with BirA was constructed by fusing in-frame a synthetic DNA-fragment encoding the artificial biotin acceptor peptide to the N-terminus of the obelin cDNA gene. The application of the fusion protein as a label in immunoassay was demonstrated. ... 

In contrast, the oxalate esters have hardly found use as chemiluminescent labels in immunoassays, despite their greater chemiluminescence efficiency. This is probably a consequence of the need to have a fluorescer in the medium with the oxalate ester to generate a measurable light signal. 

The use of enhancers will probably extend the usefulness of chemiluminescent reagents as labels in immunoassays, making chemiluminescence immunoassays a part of routine clinical analysis. 

KornickMN (1986) The use ofphycobillipro-teins as fluorescent labels in immunoassay. J Immunol Methods 92 1-13... 

Although they have not been used as labels in immunoassays, dioxetane substrates for p-galactosidase have been synthesized and characterized (B18, B23, S9). Current data are sparse, but it would appear that the enzyme is less detectable... 

F., Chemiluminescent labels in immunoassay. In Bioluminescence and Chemiluminescence Basic Chemistry and Analytical Applications (M. A. DeLuca and F. McCapra, eds.), pp. 673-679. Academic Press, New York, 1981. 

T13. Thorpe, G. H. G., Kricka, L. J., and Whitehead, T. P., Applications of enhanced luminescent quantitation of horseradish peroxidase (HRP) labels in immunoassays. Clin. Chem. (Winston-Salem, N.C. 31, 913 (abstr.) (1985). 

W4. Weeks, 1., Beheshti, I., McCapra, F., Campbell, A. K., and Woodhead, J. S., Acridinium esters as high specific-activity labels in immunoassay. Clin. Chem. (Winston-Salem, N.C.) 29, 1474-1479 (1983). 

Enzymes are one of the most commonly used labels in immunoassay. They enable measurement with a sensitivity close to that reached by radio-immunoassays but without the health hazards associated with radioactive substances. Enzyme immunoassays (El) and most importantly enzyme-linked immunosorbent... 

Kronidc,M.NH >986, TheoseofphycobiliptoteinsasflumesGem labels in Immunoassays, J, Immutu Methods... 

The effficient amplification of the sensor response i.e. the extremely high sensitivity of the enzyme electrode is the basis for the assay of enzymes used as labels in immunoassays as well as the determination of metabolites. 

Firefly, marine bacterial, Renilla and Vargula lucif-erase, and the photoprotein apoaequorin have all been employed as labels in immunoassays (Table 3). An example of the experimental design for a competitive bioluminescent immunoassay for the drug methotrexate based on a firefly luciferase label is shown in reaction [XII] ... 

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