Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Patent
1980-11-19
1983-05-24
Fagelson, Anna P.
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
436500, 436508, 436546, 436800, G01N 3348, G01N 3352, G01N 3354
Patent
active
043851266
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to techniques for diagnosing components of human blood and more particularly to a method of performing immunoassays.
BACKGROUND ART
A wide variety of techniques are known for performing immunoassays with a variety of tagging materials. Thus, immunologically active materials may be tagged with a radioactive element or a fluorescent constituent or a constituent which enters into an enzyme reaction. An immunoassay may be performed with any of these tagged materials with a variety of different immunoassay procedures. Thus a test material containing a suspected ligand may be analyzed and the ligand can be detected quantitatively by forming a complex of a tagged ligand, tagged as indicated above, with a receptor ligand and measuring the tagging constituent in the complex to deduce the quantity of the suspected ligand. The immunoassay may be performed in a number of ways, such as the well-known sandwich technique and the competitive and indirect techniques.
As used herein, the term "ligand" means any material which is capable of forming a ligand-receptor complex by means of protein interaction and the term is broad enough to include antigens and antibodies, binding proteins, haptens and hormone receptors.
The immunoassays of this invention employ at least three ligands. The three ligands are referred to as follows herein. First, there is the assay ligand which the assay has been designed to detect or measure. Second, there is the test ligand which is tagged as explained and is detected quantitatively in a complex to deduce a quantity of the assay ligand present. Thirdly, there is a receptor ligand which binds immunologically to the assay ligand and/or test ligand.
In the sandwich technique mentioned above, the assay ligand binds to immobilized receptor ligand to form a first complex. The tagged test ligand is then bound to the assay ligand in the complex to form the sandwich (the test ligand may be identical to the receptor ligand and often is), and the tagging constituent in the sandwiched ligands is detected quantitatively to deduce the quantity of assay ligand present. Detection can be performed by measuring radioactivity where the test constituent is radioactive or by measuring fluorescent light where there is a fluorescent constituent on the test ligand or spectrophotometrically where an optical density or wavelength change occurs through an enzyme reaction, or through fluorescent quenching. Detection may require separation of the sandwiched ligands from unbound ligands and this is generally done by separating the receptor ligand supported on a surface from a solution containing unbound test ligands.
The quantity of assay ligand is deduced from the quantity of test ligand detected, because the two quantities are generally directly proportional to each other in the sandwich technique. Parallel tests against known standards are employed for calibration.
The quantity of assay ligand may be deduced as as inverse proportion with the competitive technique mentioned above, where the assay ligand is contacting either simultaneously or sequentially with a known quantity of test ligand and a limiting quantity of receptor. Where the receptor and test ligands bind immunologically, the quantity of test ligand which is detected in a binary complex with the receptor is inversely proportional to the amount of assay ligand present.
In the indirect technique, a limiting amount of the test ligand will bind immunologically to either the assay ligand or the receptor ligand. The quantity of test ligand in binary complexes of test and receptor ligands is taken as an inverse measure of the quantity of assay ligand present in an equilibrium mixture of the three ligands.
The three techniques discussed above may be represented as follows where R designates the receptor ligand, A represents the assay ligand and T represents the test ligand.
______________________________________ 1. R + A .fwdarw. RA
2. RA + T .fwdarw. RAT Sandwich
1. R + A + T .fwdarw. RA + RT
simultaneous
1. R +
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Chadwick, The Lancet, Feb. 22, 1958, pp. 412-414.
Chen Janet H.
Maggio Edward T.
Rehbinder Dietrich
Fagelson Anna P.
International Diagnostic Technology, Inc.
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