Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1993-10-08
2001-02-20
Eyler, Yvonne (Department: 1642)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069100, C435S069800, C435S071100, C530S324000, C424S192100
Reexamination Certificate
active
06190885
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
This invention relates to an in vitro solid-phase competitive assay for detecting the presence of an antigenic analyte, or a binding fragment or precursor thereof in a biological sample utilizing a fusion protein of an antigenic peptide and a binding peptide, a solid support-bound antibody selectively binding the binding peptide, an antibody selectively binding the antigenic peptide, and optionally an antibody binding molecule. The present assay provides higher sensitivity and specificity than all other available assays.
2. Description of the Background
The determination of levels of different antigens in animal and human tissues took a definite turn with the development of immunoassays. The concept on which immunoassays are based is the quantitative binding of a known antigen in known quantities to an antibody in equally known quantities, and the binding of this antibody to the antigen to be used as a standard, and a comparison of this to a unknown sample comprising the antigen which will also be bound by the antibody. Antigen present in the unknown sample interfered with the initial binding of known quantities of antibody to known quantities of antigen. A key step of these assays is the separation of the bound form of the antibody or the antigen from its unbound form. Many configurations for this reaction have been proposed either as direct immunometric, competitive or displacement assays, and the like. However, to quantitate results it is in general needed to resort to hemagglutination assays, radioimmunoassays, enzyme-linked assays, and the like.
In general, in an immunoassay, a given analyte or antigen present in a animal or human tissue is or may be solubilized for mixing with the immunoassay system, and it is then compared to a solubilized known quantity of the analyte. The most common tissue analyte is blood, and more specifically serum and/or plasma from blood, but urine, cerebro-spinal fluid, different serum preparations and different animal and human tissues and fluids are also routinely assayed.
Some of the areas which have most benefited with the advent of immunoassays have been clinical chemistry, endocrinology and oncology. In endocrinology and clinical chemistry enzyme-linked assays and radioimmunoassays have been used to determine levels of hormones, proteins, and lipid metabolites, among other substances. In the field of oncology blood components, and some times tissue antigens or other molecules, indicate either the appearance of cancer or a pre-cancerous condition in animals or men. These molecules are routinely tested to monitor appearance, relapse, progression or regression of a cancer disease. These antigens or molecules are called cancer and/or tumor markers. For many years markers have been used for this purpose. An example thereof is the oncofetal antigen CEA which is used in the diagnosis of carcinomas, especially those of the colon. Other cancer markers include enzymes such as lactic dehydrogenase and alkaline phosphatase, metabolites such as prostaglandin and polyamine, proteins such as &agr;-fetoprotein and human chorionic gonadotrophin, among others. Immunoassays of these cancer markers are now applied to the diagnosis and follow up of cancer patients.
These assays generally use as standard a partially or fully purified tissue antigen. In some occasions, however, polypeptides are synthesized in the laboratory for use as antigens. The more purified the antigenic substance used as standard for the immunoassay is, the more specific and trustworthy the assay.
A set of membrane-related antigens have been used for the diagnosis of breast cancer. The antigens were originally called human mammary epithelial antigens and antibodies to them were obtained by injection of human milk fat globule (HMFG) membranes to rabbits. These were polyconal antibodies called anti-human mammary epithelial (anti-HME) antibodies. The antibodies were prepared after repeated absorptions and were found to bind breast epithelial cells selectively. The discovery of this breast epithelial system of antigens opened many new immunologic opportunities in immunohistopathology, serum assays, radioimaging and eventually immunotherapy.
The anti-HME antibodies were shown to bind to breast epithelial cell lines as well as normal breast cells, but not to fibrocytes, vascular cells, and blood cells. A special group of breast epithelial antigens (BrE-antigens) originally called human mammary epithelial antigens (HME-ntigens), are bound by absorbed anti-serum (anti-HME serum) which were created in the rabbit. These antigens were found to have 150, 70 and 45-48 Kdalton molecular weights as established by affinity chromatography, and polyacrylamide gel electrophoresis double antibody immunoprecipitation. A similar system was shown to exist in the mouse. Mouse mammary epithelial antigens may also be detected by absorbed rabbit polyclonal antisera. These antisera also identify in the mouse mammary cell membrane components having molecular weights of 150, 70 and 45-48 Kdaltons. The antigens may be detected in either normal or neoplastic mouse mammary gland. These antigens are not detected in other normal tissue cells of mice.
Other polyclonal antisera were reported to have been produced against a step-purification of HMFG antigens. These antisera are pan-epithelial in nature and reactive only against the non-penetrating glycoprotein (NPGP) complex in contrast to the original anti-HME antibodies that bind the about 45, 70 and 150 kdalton antigens. Although the anti-HME antibodies bind before absorptions to the NPGP complex, anti-HME antibody final preparations do not recognize the NPGP complex as a result of absorptions with non-breast epithelial cells to render anti-HME specific.
HME antigens may be quantitated by an immunoassay in various human breast and non-breast cell lines and in normal breast epithelial cells. High concentrations of HME antigens were found in normal breast epithelial cells and in neoplastic cells. A protease treatment of live breast epithelial cell surfaces releases most antigens therefrom. Similar results show a 48-72 hour time lapse for full reconstitution of the normal breast epithelial cell membrane after digestion.
High levels of HME antigens are found in the sera of nude mice carrying human breast tumors. These antigens can be abolished by surgical removal of the breast tumor. Anti-HME antisera were shown to have certain specificity since other transplantable human tumors such as colon, lung and melanoma, did not increase HME antigen values in mice serum.
The specificity of the assay using anti-HME antigen serum for breast tumors was tested in a nude mouse model carrying transplantable human breast tumors and compared to the specificity of an assay for sialyl transferase levels, which is also a breast cancer marker. The levels of the enzyme which is present on the breast epithelial cell membrane and the HME antigens were measured simultaneously in the sera of nude mice grafted with human breast and non-breast tumors. Breast tumor-bearing mice had elevated levels of both serum markers. However, sialytransferase levels were also elevated in non-breast tumors while HME antigens were not. Upon surgical removal of all tumors, the presence of HME antigens declined precipitously in breast tumor-bearing nude mice while sialyl transferase levels remained elevated in both breast and non-breast tumor bearing animals. This is possibly due to surgical trauma and wound healing. The higher specificity of the HME antigen assay was thus proven at least in regard to sialyl transferase, a non-specific co-habitant of the cell membrane together with HME antigens. This indicates again that most, if not all, components of the breast epithelial cell are released into circulation by breast tumors, and that assay specificity, such as is obtained with an assay utilizing HME antigens, may be required to avoid that concurrent ailments or reactions in the tumor host interfere with the values obtained from sera with markers such as sialyl transferase.
H
Ceriani Roberto L.
Larocca David J.
Peterson Jerry A.
Amzel Viviana
Arter & Hadden LLP
Cancer Research Fund of Contra Costa
Eyler Yvonne
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