Immunometric assays based

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

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

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.

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. 

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. 

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. 

Immunometric assays for TSH are available commercially as manual kit procedures or for use on automated systems. For practical reasons, nonisotopic methods dominate the market and have replaced radioactive tracer methods in most routine laboratories. The majority of immunometric TSH assays label the detection antibody with chemiluminescent labelled molecules other labels include peroxidase or alkaline phosphatase and sensitive photo-metric and fluorescenri molecules. Other assays are based on the use of fluorescent labels using europium chelates chemiluminescent compounds such as acri-dinium esters or ruthenium or bioluminescent molecules such as recombinant aequorin. ... 

Third generation immunometric assays have been developed for TSH measurements that have enhanced precision at even lower detection limits. Most of these assays use chemiluminescence-based technologies and have a functional detection limit of less than 0.02 mlU/L. Fourth generation assays having a functional detection limit of 0.001 to 0.002 mlU/L range also have been developed. ... 

Tg antibodies are directed against the Tg protein, a major constituent of thyroid colloid. Several different techniques have been used to detect and quantify TgAb in peripheral blood. These include passive hemagglutination, the agar gel diffusion precipitin technique, immunofluorescence of tissue sections, enzyme-linked immunosorbent assay (ELISA), radioassay techniques, and chemiluminescence-based immunometric assays. 

Moore P> Layte K, Aslam M, Martin JK. One-step immunometric assay for thyroid stimulating hormone (TSH) based on enhanced luminescence. Clin Chem 1989 35 1149. 

M9. Mashiter, K., Love, C., Loizou, M., Aslam, M., Allen, G. J., and Holian, J., Development of a simultaneous incubation two-site immunometric assay for follicle stimulating hormone (FSH) based on enhanced luminescence. Clin. Chem. (Winston-Salem, N.C.) 33, 892 (abstr.) (1987). 

The first automatic immunoanalyzers used fluorescent labels. These provided automation and short turn-around times, but due to a fairly high nonspecific background the sensitivity of assays was not improved over that obtained by RIA. Introduction of time-resolved fluorescence facilitated the development of assays with substantially reduced detection limit [1], here called improved sensitivity. This was based on reduction of the background, which facilitated the use of a large excess of label in sandwich-type immunometric assays and a much extended measuring range [3-5]. Other nomadioactive reporter molecules, e.g., luminescent labels or enzymes combined with luminescent or fluorescent substrates, can provide similar sensitivity of assays [2]. However, time-resolved fluorometry is unique in facilitating development of fully automated in-house assays, and it has therefore become widely used in research laboratories. 

The majority of assay methods for excreted low molecular weight proteins are based in immunometric methods (gel immunodiffusion, nephelometry or ELISA) and the antibodies do not cross-react with homologous proteins in animal urine (the exception is p2 -microglobulin, for which a rat-specific latex... 

The development of various EIA procedures has led to confusing terminology and classifications, which are often misleading concerning their fundamental features and tend to obscure their relative merits. Not surprisingly, many comparative studies produced inconclusive results. The frequent comparison of the relative merits of radioactive and enzyme labels, as evaluated in RIA by saturation analysis and in ELISA by immunometric analysis, is basically faulty since the underlying principles of these assays are different (Ekins, 1980). Here, EIA will be classified according to their essential differences to expose the inherent limitations and potentials of each assay EIA are based either on Activity Amplification (AA) or Activity Modulation (AM). 

The assay format for C-peptide was based on the two-step method and the two-site immunometric principle using two MoAbs, which recognize different epitopes. 

The first quantitative immunoassays were based on the use of radioactive reporter molecules, mainly and " C. These radioimmunoassays (RIAs) have gradually been replaced by nomadioactive reporter molecules or labels, e.g., enzymes, fluorescent, and luminescent molecules and combinations of these. The nonradio-active labels facilitated development of automated immunoassays, and they also contributed to the design of assays with substantially reduced detection limits. However, nomadioactive assay have an advantage only in immunometric sandwich assays in inhibition assays, the use on nonradioactive labels has not provided any improvement in assay sensitivity as compared to RIA [1]. The development in immunoassay technology has been reviewed by Ekins [2]. 

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