Idiometric assay: noncompetitive immunoassay for small molecules typified by the measurement of estradiol in serum

Abstract

We report here a novel noncompetitive immunoassay applicable to the measurement of small-molecular-mass compounds and typified by the direct measurement of estradiol in serum. Two types of anti-idiotypic antibody recognize different epitopes within the hypervariable region of the specific primary antibody (e.g., anti-estradiol). The first anti-idiotype (betatype) recognizes an epitope at the unoccupied binding site, which is masked in the presence of the analyte (e.g., estradiol). The second (alphatype) recognizes an epitope close to the binding site and is unaffected by the presence or absence of the analyte, but is sterically hindered from binding to the primary antibody in the presence of the betatype. The use of these matched antibodies (primary antibody, alphatype, and betatype) has enabled the development of a method for determining antibody occupancy that is not based on a conventional two-site assay. An excess amount of purified monoclonal antibody against estradiol is immobilized onto the walls of microtiter wells. After the capture of analyte, the unoccupied antibody sites are blocked by the addition of an excess amount of betatype. Subsequently, analyte occupancy is determined by the addition of excess europium-labeled alphatype, incubation, washing, and time-resolved fluorometry. The method demonstrates good sensitivity, precision, and comparability with alternative competitive immunoassays.

Antivascular Treatment of Solid Melanoma Tumors with Bacteriochlorophyll-serine-based Photodynamic Therapy¶

“…Determination of vascular leakage of Eu-IgG. To determine PDTinduced vascular leakage, we measured the release of fluorescently labeled mouse IgG (Sigma Chemical Co., St. Louis, MO) labeled with Europium-diethylenetriamin-pentaacetate (Eu-DTPA) Europium immunoglobulin G (Europium-IgG) (38) from the blood stream to the tumor tissue. Mice implanted with two tumors, one on each flank were i.v.-injected with Bchl-Ser (15 mg/kg); only one tumor was illuminated while the tumor on the contralateral side was kept in the dark (dark control).…”

Oestradiol assays: fitness for purpose?

“…11 Radioimmunoassays (RIA) became available as in-house systems in the early 1970s, with commercial assays following quite soon after. Other types of ligand-binding assay and even non-competitive format 'idiometric' assays 12 have been investigated, but none of these has been a serious contender to the basic competitive, limited reagent, labelled antigen, assay format.…”

Ways to Reach Lower Detection Limits of Lateral Flow Immunoassays

“…Its solutions for various types of immunoassay are summarized in the reviews of Fan and He [79] and Liu et al [80]. Unfortunately, many of these approaches, such as idiometric assay [81] and immunoassay using anti-metatype antibodies [82] require the production of antibodies not simply against the target analyte but against more complex antigenic structures, which limits their widespread use. A more universal idea is to use quenching of fluorescence caused approaching between donor and acceptor in the binding zone of the test strip.…”