Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-09-13
2002-12-31
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
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Reexamination Certificate
active
06500629
ABSTRACT:
BACKGROUND OF THE INVENTION
There are a number of assay systems available for detection and quantitation of analytes, particularly analytes of biological interest. Current assay systems include enzyme immunoassay (EIA), radioimmunoassay (RIA), and enzyme linked immunosorbent assay (ELISA). Among the analytes frequently assayed with such systems are: 1) hormones, such as human chorionic gonadotropin (hCG), frequently assayed in urine as a marker of human pregnancy; 2) antigens, particularly antigens specific to bacterial, viral, and protozoan pathogens, such as Streptococcus and hepatitis virus; 3) antibodies, particularly antibodies induced as a result of infection with pathogens, such as antibody to the bacterium
Heliobacter pylori
and to human immunodeficiency virus (HIV); 4) enzymes, such as aspartite aminotransferase, lactate dehydrogenase, alkaline phosphatase, and glutamate dehydrogenase, frequently assayed as indicators of physiological function and tissue damage; 5) other proteins, such as hemoglobin, frequently assayed in determinations of fecal occult blood, an early indicator of gastrointestinal disorders such as colon cancer; 6) drugs, both therapeutic drugs, such as antibiotics, tranquilizers and anticonvulsants, and illegal drugs of abuse, such as cocaine, heroine, and marijuana; and 7) environmental pollutants such as pesticides and aromatic hydrocarbons and vitamins.
Such systems are frequently used by physicians and medical technicians for rapid in-office diagnosis and therapeutic monitoring of a variety of conditions and disorders. They are also increasingly used by patients themselves for at-home or on-site monitoring of such conditions and disorders.
Among the most popular of such assay systems are immunoassays, which depend on the specific interaction between an antigen or hapten and a corresponding antibody. The use of immunoassays as a means of testing for the presence and/or amount of clinically important molecules has been known for some time. As early as 1956, J. M. Singer reported the use of an immune-based latex agglutination test for detecting a factor associated with rheumatoid arthritis (Singer et al, 1956).
Development of the first radioimmunoassay by Rosalyn Yalow and Sol Berson (1959) set the stage for measurement of a wide variety of hormones in biological fluids by binding the hormone specifically and with high affinity to antibodies developed in animals against the hormone in question. The assay developed by Drs. Yalow and Berson employed antibodies formed against the protein hormone, insulin, and utilized a radiolabeled form of insulin as the marker, or “reporter” hormone. Antibodies became a useful way to “capture” a specific hormone from biological fluids and under conditions of constant antibody concentration and with some easily detected source of labeled hormone (usually radioactively labeled; hence the name “radioimmunoassay”) the amount of hormone “captured” from the biological fluid could be quantified by comparison to known concentrations of the hormone in similar conditions. In practice of the art, known amounts of (unlabeled) hormone, (insulin in the example) were allowed to compete for binding to the antibody with a known and fixed amount of I
131
labeled insulin. The radiolabeled form of hormone, and the amount of antibody were held constant while the amount of unlabeled hormone was varied. This was the basis of a “standard curve” from which the amount of radioactive label that bound to the antibody varied inversely with the amount of unlabeled hormone. Comparison of the mass of unlabeled hormone required to displace a given amount of labeled hormone could then be used to estimate mass of an sample hormone. Separation of the fractions which were unreacted with the antibody (unbound) was carried out by a variety of chemical separation methods. In the original teaching of Yalow and Berson (1959), separation of the antibody-bound fractions of insulin from the unbound (free) fractions of insulin was carried by electrophoresis. Subsequent to their report, many means of separating bound from free fractions have been utilized, including column chromatography, salt or organic solvent precipitation of the protein (antibody), double antibody (in which the gamma globulin fraction of the species immunized against the hormone is then introduced to a different species to create an anti-antibody, or second antibody, and solid phase, in which the antibody is held by electrostatic forces to a solid interphase such as the inner wall of a test tube, flat disc, or elongate stick (dipstick) and separation of bound from free requires simple physical separation of the solid phase from the liquid phase containing the free fractions. A variant of the technique of radioimmunoassay involved coupling small, non-immunogenic molecules to larger, highly immunogenic molecules, such as bovine serum albumen (BSA), thyroglobulin (TG), or keyhole limpet hemocyanin (KLH) and stimulation of antibodies that recognized the smaller, non-immunogenic, portion of the hapten molecule. This modification of the technique permitted quantification of small hormones, such as steroids and prostaglandins.
While radioimmunoassay is a very useful tool for conducting research and for certain clinical applications, it has several drawbacks as far as practical management of endocrine or other hormonal states. A major drawback is the use of an antibody as a “capture protein.” Development of polyclonal antibodies is accomplished by administering the hormone to an animal that regards it as foreign and develops antibodies against it. The process is very much trial and error and involves the use of a number of animals and screening of the antibody before determination of its usefulness. Once an ideal polyclonal antibody preparation has been obtained, the animal's plasma must be harvested and husbanded carefully, for once the animal dies, the supply of that particular antibody is lost forever.
The act of mounting an immune attack against a foreign protein and producing antibodies is actually a mixture, or collection of antibodies (hence the term “polyclonal antibody”), each of which is directed against a particular amino acid sequence called an epitope. A refinement of this process involves the production of monoclonal antibodies. Monoclonal antibodies are derived by collecting individual spleen cells from animals immunized by administration of a foreign protein and culturing the lymphocytes in vitro. The cells are then screened to determine their binding characteristics, and those cells that possess appropriate binding are then cloned and maintained as an antibody-specific, continuous cell line. Thus, once appropriate cell cultures are obtained, they may be kept essentially indefinitely, thereby obviating one of the negative aspects of polyclonal antibodies.
However, monoclonal antibodies also have some drawbacks. For one thing, they are so specific as to be a detriment in some cases. Monoclonal antibodies are directed against amino acid sequences (epitopes) that are often common features of the tertiary structure of proteins. In this case the monoclonal antibodies are really not specific as one might believe at first. This drawback can be overcome by very stringent screening and validation of the assays utilizing monoclonal antibodies, but greater effort is often required. Additionally, monoclonal antibodies tend to be monovalent, which may restrict hierarchical or sandwich type coupling to other molecules for the purpose of separation or of amplification of the reporter signal.
Immunoassays fall into two principal categories: “sandwich” and “competitive,” according to the nature of the antigen-antibody complex to be detected and the sequence of reactions required to produce that complex. Generally, the sandwich immunoassay calls for mixing the sample that may contain the analyte to be assayed with antibodies to the analyte. These antibodies are mobile and typically are linked to a detectable label or a disclosing reagent, such as dyed latex or a radioisotope. This mixture is t
Cleaver Brian D.
Green Mike L.
Chin Christopher L.
Cook Lisa V
Equitech Laboratories, Inc.
Saliwanchik Lloyd & Saliwanchik
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