MXPA97005161A - Protein "a" as can diagnosis - Google Patents

Abstract

The presence of protein A in an abnormally large amount in a sample, such as blood, of an individual is diagnostic of primary cancer or metastasis therein. The presence of protein A is more easily detected by means of the immunological reaction of it with specific antibodies. A preferred method for detecting the presence of protein A in samples is by means of a "sandwich" type assay using two antibodies with different epitope specifications for the protein

Description

PROTEIN AS A DIAGNOSIS OF CANCER Background of the Invention The diagnosis of cancer in individuals remains somewhat difficult to perform. Although some diagnostic markers are available that can be studied from blood or tissue samples, for example the Carcinoembryonic Antigen (CEA), Alpha-Fetoprotein (AFP) or the Prostate Specific Antigen (PSA), the trials that use these markers have not predicted markedly, to date, the presence of cancer in these individuals, as verified by other clinical diagnoses. The sensitivity and specificity of these trials have been disappointingly low. Clinical assessments (eg, palpations, X-rays, mammograms, biopsies) that have remained accepted methods for diagnosing cancer are time-consuming and labor-intensive. Thus, a marker, preferably present in a biological sample of an individual, such as blood, which is predictive of the presence of cancer in the individual is needed. In particular, the existence of a marker and an assay is needed to measure the presence and quantity of this marker for individuals who have cancer at an early stage. If such a diagnostic test were available, early treatment with beneficial results would be more likely than at the present time. It is an object of this invention to provide methods for detecting the presence of primary or metastatic cancer in an individual that involve the detection of a cancer diagnostic protein. It is also an object of this invention to provide compositions of matter that can be used to detect the presence of primary or metastatic cancer in an individual. SUMMARY OF THE INVENTION This invention relates to methods of detecting cancer in an individual by the results of a test of a biological sample, such as a blood sample, of the individual. In this assay, the sample is incubated with at least one immunoreactive antibody with a cancer diagnostic protein that may be present in the biological sample. The immunoconjugates that are formed in the reaction diagnostic protein of cancer: antibody are detected. The presence of an abnormally high concentration of these immunoconjugates indicates that the individual from whom the sample was taken has primary or metastatic cancer. In a preferred embodiment of this invention, the cancer diagnostic protein is protein A and the sample is incubated in a sandwich assay for protein A. An immunoreactive antibody with protein A is attached to a solid support. The sample is allowed to immunoreact with the bound antibody and with a second antibody that is immunoreactive with another protein A region (i.e., a region other than the immunoreactive region with the antibody bound to the solid support). The resulting complex of two antibodies-protein A thus formed forms a sandwich. The amount of bound second antibody is detected. This amount of second antibody detected is directly proportional to the amount of protein A bound. An abnormally large amount of second antibody detected is indicative of the presence of primary or metastatic cancer that is being detected by testing the biological sample. Another embodiment of this invention is a test it containing one or more antibodies for use in the cancer diagnostic protein assay (e.g., protein A). One of the antibodies is immunoreactive with an epitopic region of the cancer diagnostic protein and, if a second antibody is included, the second anti-body is immunoreactive with an epitopic region of the cancer diagnostic protein separated from the epitopic region that is immunoreactive with the first antibody. In a preferred embodiment of the assay kit, there are two antibodies that are immunoreactive with two epitope regions of protein A. One of the antibodies binds to a solid support, such as the walls and bottoms of the wells of a microtitre plate. The other antibody has a detection label attached thereto. Yet another embodiment of this invention is one or more antibodies that immunoreact with a cancer diagnostic protein, ie, protein A. These antibodies are used to detect or assess the presence of the cancer diagnostic protein. The titration can be performed using biological samples, such as blood. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graphic representation of the chromatographic separation of soluble protein A: Fig. IA is an optical scan of the As form of purified protein A on a liquid chromatography gel filtration column. High pressure (HPLC); FIG. IB is an optical scan of the As purified by rechromatograted HPLC on an anion exchange column of DEAE-cellulose with a linear saline gradient (dotted line) to confirm the efficiency of the purification process. Fig. 2 is a photographic representation of the purification of the A, and As forms of protein A by SDS-PAGE. The gel was dyed with silver. Band 1 is A ,, purified; band 2 is purified As; band 3 shows molecular weight patterns. Fig. 3 is a graphical representation of GTP-PNP analog binding of GTP-PNP by soluble and membrane-bound forms of protein A in the presence of adenosine nucleotides. Fig. 4 is a graphical representation of the ATPase activity of As and A ,,, in the presence of GTP? S. Fig. 5 is a scatter plot representing the concentration of protein A in blood for control individuals and for individuals who have been diagnosed with metastatic breast cancer. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to methods and compositions for diagnosing the presence of primary or metastatic cancer in individuals. The primary cancer is a cancerous growth confined to a specific anatomical region of the organism or is composed of cells that formed the initial cancerous lesion. Metastatic cancer is a cancerous growth that has spread from an original primary site in the body through the blood or lymphatic system or both and grows in a site separate from the primary tumor; it is also a recurrent disease disseminated secondary to the treatment of a primary tumor. The primary or metastatic cancer that can be diagnosed using the methods and compositions of this invention is any of a wide variety of cancers. In this list of cancers detectable by the methods and compositions of this invention include breast cancer, prostate cancer, liver cancer, lung cancer, colorectal cancer, gastric tissue cancer, pancreatic cancer, bladder cancer, head and neck cancer, endometrial cancer, parotid cancer, cholangiocancer, kidney or kidney cancer, cervical cancer, thyroid cancer, brain cancer, cancer mouth, uterine cancer, abdominal cancer, tongue cancer, lip cancer, anal cancer, pelvic cancer, inguinal cancer, cancer of the penis, cancer of the costal wall, cancer of the fallopian tube Fallopian, POEMS, lymphoma, leukemia, multiple myeloma, melanoma and various sarcomas. These cancers and, most likely, other types of cancer, can be detected at various stages of their progression, including stage 1 and stage 2. Detection of the presence of these cancers can be distinguished from the presence of benign tumors or of the absence of cancer in individuals using the methods and compositions of this invention. The methods of this invention preferably involve the use of antibodies that are immunoreactive with a cancer diagnostic protein. The preferred cancer diagnostic protein of this invention is protein A. After incubation of one or more of the antibodies with a biological sample from the individual, the presence of an abnormally large amount of immunocontained antibody-protein A indicates the presence of primary or metastatic cancer in the individual. Blood is a frequently used and preferred biological sample. When the blood is the biological sample, the preferred constituent of the blood being analyzed is plasma or serum, more preferably serum. Since blood or other organic fluids are preferred biological samples, the immunoreactive cancer diagnostic protein, for example protein A, is detected in a liquid medium. When the blood is the biological sample, the liquid medium is circulating in the organism. Therefore, the methods of this invention are not based on an analysis of localized material, for example a biopsy, but, rather, they can be employed with a readily obtainable biological sample, such as serum or blood plasma. Even when such a blood sample is used, the variety of cancers that can be detected is quite extensive. In particular embodiments of the invention, the presence of a concentration of protein A in a biological sample of a particular individual that is more than twice the concentration of protein A found in similar samples of individuals without cancer is indicative of the presence of cancer in that particular individual. In other words, if you take biological samples of the same size from individuals that are cancer-free and determine the average amount of protein A in these samples, an individual is predicted to have cancer if a biological sample of the same size as that individual has cancer. more than twice the amount of protein A in it that is determined as the average amount of protein A for cancer-free individuals. In these particular embodiments, the factor of 2.0 is considered to be a threshold value. Individuals are predicted to have cancer if protein A concentrations in biological samples of them have more than 2.0 times the average concentration of protein A for cancer-free individuals, that is, if the ratio of protein A concentration of the sample of the individual studied with respect to the average protein A concentration of cancer-free individuals is greater than 2.0. The reasons for the protein A concentration of the individual studied at the mean protein A concentration of cancer-free individuals less than 2.0 indicate that the individual studied does not have cancer. The threshold value of 2.0 is a particularly preferred value. Other threshold values can be established to predict the presence of cancer in an individual. These threshold values will depend on the accuracy and reproducibility of the specific cancer diagnostic assay, as well as the predictive reliability sought for the trial. The actual amount or concentration of protein A in a biological sample of an individual may have a diagnostic or prognostic value. By determining the amount of protein A over a period of time for an individual, it is possible to monitor the progression of the cancer (by increasing the amount of protein A) or the regression of the cancer, eg. as a result of therapy (by decreasing the amount of protein A). The amount of protein A detected can be significant as an indicator of the presence and vigor of the cancerous growth. The amount of protein A is monitored relative to a baseline value for that individual or compared to the average amount of protein A in similar biological samples from cancer-free individuals. When the amount of protein A is greater than a particular value, established by the test monitor, the individual is considered to have cancer. The actual amounts of protein A above this established value indicate the severity of the cancer and smaller amounts of protein A, with or without therapeutic treatments, indicate a decrease in the severity of the cancer. In other specific embodiments of the invention, the presence of more than about 10 ng of protein A in blood is predictive of metastatic cancer. In these methods, assays for metastatic cancer are also based on measurements of the amount of protein A in biological samples (eg, fluid samples such as plasma or blood serum, urine, etc.) of an individual. Again, the amount of protein A in the biological sample is indicative of the presence or absence of metastatic cancer in the individual from whom the sample was obtained. An abnormally large amount of protein A indicates the presence of metastatic cancer; a normal amount of protein A indicates that the individual does not have metastatic cancer. The utility of these trials is readily apparent: a relatively simple trial predicts the presence or absence of primary or metastatic cancer.

The artisan knows and has a variety of techniques to study the amount of protein A in a biological sample. These techniques include the isolation and quantification of protein A in the sample by solvation and centrifugation procedures, by column chromatography or electrophoretic gel separation methods, by filtration procedures, by enzymatic recognition procedures or by methods of binding with molecules that recognize and bind to protein A with specificity, such as affinity chromatography or immunoassay. Said specific binding methods can also be used to detect and quantify protein A in si tu in biological samples by assessing the volume or area of distribution of the specific binding molecule in the sample. A preferable technique for studying the amount of protein A in a biological sample is immunological recognition with antibodies that bind specifically to protein A or particular epitopes of protein A. Said antibodies are compositions of the present invention. These antibodies selectively recognize determinants of protein A and bind to these determinants with high affinity. The antibodies can be used alone as affinity immobilization or as label binding moieties in methods such as Western blotting or electrophoretic distribution analysis, or multiply to bind different protein A determinants or epitopes such as assays in sandwich. These antibodies may have substances that act as labels attached to them to facilitate identification after binding of the antibody to protein A. The antibodies of this invention bind to protein A with specificity, so that protein A or specific epitopes of protein A can be detected with particularity. The antibodies that can be used in this invention are reactive with protein A. The term "antibody" is also intended to encompass both polyclonal and monoclonal antibodies. The term "antibody" is intended to encompass mixtures of more than one antibody reactive with protein A (eg, a cocktail of different types of monoclonal antibodies reactive with protein A). The term "antibody" is also intended to include whole antibodies, biologically functional fragments thereof, single strands or single strand fragments with protein A binding properties and chimeric antibodies consisting of portions of more than one species, bifunctional antibodies, etc. The fragments of biologically functional antibodies that can be used are those fragments sufficient for the binding of the antibody fragment to protein A. The chimeric antibodies can consist of portions derived from two different species (e.g., human constant region and variable region or murine union). Portions derived from two different species can be linked together chemically by conventional techniques or can be prepared as fusion proteins using genetic engineering techniques. In addition, the DNA encoding the proteins of both the light chain and the heavy chain portions of the chimeric antibody can be expressed together as fusion proteins. It has been determined that particular intracellular molecules, called proteins A, are intimately involved in the regulation of the signal transduction system related to inositol. In this system, proteins A function by activating phospholipase C (PL-C) to generate the second messengers inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DG). By stimulating a membrane-bound receptor, a protein A binds to GTP to form an intermediate that works by activating the PL-C. When the GTP of the intermediate is hydrolysed to GDP, the activation of PL-C ends. The proteins A are, consequently, important G-type signal transduction molecules critical for the proper functioning of the metabolic pathway of cellular inositol. These signal transduction molecules are useful in the production of the antibodies used in the above methods of the present invention. The signal transduction molecules used to produce the antibodies are isolated and purified proteins A and epitope fragments of protein A. The cancer diagnostic proteins of the invention, for example protein A, may also be useful in immunotherapy. When these proteins are introduced into the blood or lymphatic system, they can stimulate the immune system to give an answer, that is, to recognize the presence of cancer in the host and respond to it. In this way, by introducing these cancer diagnostic proteins into the bloodstream or into the lymphatic system of the individual from external sources, the immune system can be stimulated to generate immunosuppressive activity and eradicate the cancer. In addition, the antibodies of this invention may have therapeutic value. When administered to an individual, they can immunoreact with the cancer diagnostic protein, e.g. protein A, and thereby initiate a greater immune system response, for example by stimulating the production of T cells, to kill the cancer cells present in them. the individual. The term "protein A" was originally used to describe an elongated photoreceptor protein of approximately 20 kilodaltons in molecular weight based on the electrophoretic mobility of this protein through a gel under reducing conditions. From an analysis of the protein as an expressed product of the known protein A gene, the molecular weight of protein A is 26 kilodaltons. Better extraction and separation methods combined with the preliminary sequence data on the separated forms indicate that the entity previously referred to as protein A may consist of at least two structurally and functionally related proteins: one bound to membranes and one soluble. On this basis, the terminology used reflects the assumed in vivo state: A,., Bound to membranes (20 kD) and As, soluble (19 kD), again on the basis of electrophoretic mobility under reducing conditions. These A proteins include the amino acid sequences for the N-terminal regions indicated in the Sequence List as: GNSKSGALSKEILEELQ (SEQ ID NO: 1) (AJ and MGNSKSGALSKEILEELQ (SEQ ID NO: 2) (AJ) Other characterizations of the molecules related to protein A are that they consist of a single polypeptide chain with a significantly hydrophobic region. They also have the ability to bind and hydrolyze adenosine triphosphate and guanosine nucleotides and have the ability to activate phospholipase C, phospholipase D and possibly also phospholipase A2 in the presence of GTP. elongated outer segments (ROS) of mammalian (bovine) and amphibian (frog) of the photoreceptor cells of the eye by extraction, centrifugation, chromatography and other protein purification techniques known to those skilled in the art. functional similar or identical to proteins A have been isolated from various other vertebrate and invertebrate tissues. These discoveries indicate that the structure of proteins A has been conserved throughout evolution. Proteins A are quite labile in aqueous solution, but can be significantly stabilized if they are available in aqueous solutions containing a non-ionic surfactant. They have a molecular weight in the range of 19 to 20 kD, as deduced by comparison with molecular weight patterns during electrophoretic separations under reducing conditions. Next, preferred methods of isolation of the native protein are described in detail. Good purification results have been achieved using filters with molecular weight cuts in the range of 10 kD to 30 kD. The A proteins, various truncated or mutein analogues thereof, and fusion proteins consisting of a protein A and domains of other proteins can be produced by various synthetic and biosynthetic means. For example, an appropriate host cell, such as a culture of microorganisms, yeasts or eukaryotic cells, can be engineered to express a protein A, or a portion or analogue thereof. This can be accomplished by currently well established recombinant DNA technologies known to those skilled in the art. The recombinant method can include the isolation or synthesis of the gene encoding a protein A, a portion or analogue thereof and the integration of that gene into a plasmid. The amino acid sequences of proteins A can be easily established. The Sequence List establishes the N-terminal amino acid sequence of two forms of protein A (A ,, and AJ as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.) Gene synthesis from synthetic oligonucleotides and Known mutagenesis techniques provide the technologies for preparing an array of truncated analogs, protein A forms and fusion proteins consisting of protein A or an antibody-binding domain thereof.The production of such materials can also include the transformation of an appropriate host cell with a vector harboring the recombinant DNA that includes the gene encoding protein A or a portion or analogue thereof, the culture of that transformed host cell and the isolation of the expressed protein. protein-rich A producible as described herein, the recombinant production of the native form and various portions and analogues of the isma is within current knowledge in the art. Alternatively, at least portions of the protein can be produced synthetically by chemical linkage of amino acids in the correct sequence. The isolated proteins A, or a portion or analogue thereof, can be used as antigens to produce antibodies that are useful for detecting protein A in fluid samples of individuals, thus studying the presence of primary or metastatic cancer. The antibodies can be part of a polyclonal antiserum, or their binding portions of these antibodies, produced against protein A, and which show reacting with protein A or with its analogs, fragments or a particular epitope on A ,, or As. The antibodies can be polyclonal or monoclonal antibodies produced by methods known per se. The antibodies are preferably selected so that they do not cross-react with other cellular components. The antibodies can be of any class and subclass, as determined by the Ouchterlony double diffusion assay. Antibodies of the IgG class are preferred. Alternatively, antibodies that recognize protein A can be synthesized by biosynthetic or recombinant means, either in whole or in part. In addition, the antibodies can bind to other functional molecules such as toxins, fluorescent or absorption dyes, enzymes or radioactive labels. In preferred embodiments, the antibodies bind to biotin molecules, which have a particularly strong avidity for avidin or streptavidin, which, in turn, can bind to fluorescent, absorption or radioactive labels. The bound antibodies can be used to detect protein A in biological samples and thus study primary or metastatic cancer. The antibody-marker complex can be prepared by chemical binding or by recombinant DNA techniques if the marker is protein. The antibodies can also be labeled with a reagent that allows the monitoring or imaging of the antibody immediately after its administration to a patient. Labeling may be, for example, a radioisotope such as 125 I or 99raTc, both of which may be followed by extracorporeal imaging by radiation detection means such as a gamma scintillation chamber. Alternatively, the antibody can be labeled with a non-radioactive paramagnetic contrast agent capable of detecting in MRI systems. In such systems, a strong magnetic field is used to align the nuclear spin vectors of the atoms in a patient's organism. The field is then altered and an image of the patient is read as a return of the nuclei to their equilibrium alignments. In the present invention, antibodies can be attached to paramagnetic materials such as gadolinium, cobalt, nickel, manganese or iron complexes to form conjugated diagnostic contrast reagents whose image is extracorporeally followed with an MRI system. If the antibodies are not bound to functional markers, the antibody-protein A conjugates can still be detected using standard biochemical techniques to recover immunoprecipitates, such as centrifugation. Anti-protein A monoclonal antibodies can be obtained from hybridoma cell lines formed after fusing mouse myeloma cells with spleen cells from mice previously immunized with protein A that has been purified, for example, from bovine ROS. The immunogen may alternatively be a derivative of protein A, or an analog or portion thereof, produced in vi tro according to known manual or mechanical methods of peptide synthesis. Alternatively, the immunogen (protein A) can be synthesized by biosynthetic means using recombinant DNA technologies known to those skilled in the art. Mice whose spleen cells are selected for fusion are preferably of a genetically defined line, such as Balb / C. The myeloma cells used in the fusion are from a mammalian antibody producing cell line, but, more preferably, they are from a mouse cell line such as, for example, NS-1. The monoclonal antibodies can be obtained from the ascitic fluid of mice injected with the fusion product. In preferred embodiments of the invention, an immunogen for the production of polyclonal or monoclonal antibodies is a peptide with an amino acid sequence of about 16 amino acids taken from the known amino acid sequence of protein A. This immunogen is a peptide of 10-18 amino acids in length. In particularly preferred embodiments, the immunogen is the carboxyl terminus of 14 amino acids or 16 amino acids of protein A, the peptide that contains amino acids 60-71 of protein A, the peptide that contains amino acids 142 to 158 of protein A or the peptide that contains amino acids 158-170 of protein A. When this immunogen is used to produce antibodies for incorporation into a sandwich assay, a second immunogen of protein A other than the peptide of about 16 amino acids of the first immunogen is chosen. Another technique for obtaining antibodies that immunoreact with the second immunogen of protein A is to immunize an animal with intact protein A and select antibodies that immunoreact with an epitope of protein A other than the first immunogen. The production of two groups of antibodies with immunoreaction properties with different epitopes ensures that sandwich assays employing these antibodies will not be impeded by competition of the antibodies for the same antigenic site. This characteristic increases the sensitivity of the assay for the detection of the actual amount of protein A present in the sample. Polyclonal or monoclonal antibodies thus produced by known methods are specific for protein A and, therefore, are particularly useful for studying proteins A. In the present invention, intact proteins A or detectable fragments of proteins A can be studied by described methods. The essential characteristic of the intact protein or of the detectable fragments thereof is the ability of these proteins or peptides to be detectable, ie, to immunoreact with immobilization or detection antibodies. The formation of protein complexes or fragments with the antibodies and detection of these complexes is all that is required. Said detectable complexes may be formed with protein A fragments. This invention includes a particular method for detecting the amount of protein A in a biological sample, such as serum or blood plasma. In this method, a first antibody, which binds to a first epitope on protein A, is adhered to a solid support. The first adhered antibody is brought into contact with the biological sample to be studied. Either simultaneously or sequentially thereafter, a second labeled antibody, which binds to a second epitope on protein A, is added to the mixture of the first antibody-biological sample, thus forming a first immunocomplex first antibody: protein A: second antibody bound to the solid support. This second antibody is bound to a detectable label. Any unbound second antibody is separated and the presence and, if desired, the amount of the label is then detected, its presence and amount of the presence and amount of protein A in the sample being indicative. An abnormally large amount of detectable protein A, by said assay, indicates that the individual from whom the biological sample was obtained has a high probability of having primary or metastatic cancer. In other words, this assay is diagnostic for primary or metastatic cancer. Test kits are also embodiments of this invention. These test kits contain one or more of the polyclonal or monoclonal antibodies that are needed to perform assays for the cancer diagnostic protein, for example protein A, which may be present in biological samples, such as blood, obtained from individuals . These test kits may also contain solid supports, such as microtiter trays, to carry out the tests. The instructions for testing for protein A may also be included in the kits. If desired, an identification tag can be attached to an antibody from the test kits. In preferred embodiments of the test kits, antibodies are provided that allow sandwich assays when the antigen is protein A. In particularly preferred embodiments of the invention, one of the antibodies in the sandwich is not labeled and adheres to a solid support. The other antibody has a label attached thereto for detection purposes. The following examples further describe the nature of the invention, without limiting its scope. EXAMPLE 1 Purification of soluble protein A (AJ and membrane bound (AJ) Proteins A were isolated from cow's eye retinas essentially as described by Schmidt et al (J. Biol. Chem., 252: 14333-14336 (1987)) Bovine eyes (cow or calf) were obtained from a local abattoir within two hours after slaughter, the bovine eyes were kept on ice and in darkness for 30 to 60 minutes, the retinas were dissected and placed in buffer A (NaCl). 130 mM, 20 mM Tris-HCl, pH 7.0; 1 ml per calf retina or 2 ml per cow's retina). By means of soft and repeated inversions of the vessel, large quantities of ROS were released into the buffer. The mixture was poured through a Buchler funnel to separate the retinas. The filtrate was allowed to settle in a conical bottom tube on ice for 5 minutes, allowing the coarse particulate to settle and separate from the ROS suspension. It was seen, by means of microscopic examination in a hemocytometer, that the supernatant consisted of more than 95% ROS. The ROS were broken with the shear created by repeatedly bringing the suspension into the syringe and forcing it outside against the wall of the container. To separate the particulate and aqueous fractions, the suspension was centrifuged at 10,000 to 12,000 x g for 20 minutes at 4 ° C. The granule containing the ROS membranes was washed once with a volume of buffer A equal to that of the supernatant removed. The resulting granule was resuspended in 3 to 6 ml of T buffer (Tween 20/80 (1: 1) (0.05%) in double distilled water) and rotated at 15,000 x g for 45 minutes. The above manipulations were carried out under weak red light. Both protein A solutions (supernatants containing soluble and membrane bound protein A, respectively) were filtered through Centricon 30 microconcentrators (30 kD molecular weight cutoff, Amicon Corporation), with centrifugation at 5,000 xg in a refrigerated centrifuge. -gives. The ultrafiltrates were then concentrated and dialysed in Centricon 10 (molecular weight cut-off of 10 kD) at 5,000 x g. The retenidos contained proteins of 10 kD to 30 kD, with average concentrations of 100 to 200 μg / ml for soluble protein A (AJ and 30 to 100 μg / ml for the membrane bound (AJ, reduced to a volume of 0.5 The purification of As resulted in a 320-fold enrichment and A ,, was purified 20 times.If the soluble protein A solution had to be stored overnight before its concentration and use, 1 was added: 5 buffer A with Tween 20/80 (1: 1) 0.05% to minimize aggregation of the proteins in the concentrated solution Proteins A, purified by ultrafiltration as described above, were still purified for sequence analysis by HPLC on a Bio-Sil SEC-125 column in buffer A (AJ or T buffer (AJ) The elution was isocratic The collected peaks were concentrated, dialyzed against water and lyophilized before analysis, in order to confirm the purity of the the protein A soluble used in the experiments, the As, purified by ultrafiltration as described, was carried to a size exclusion column of HPLC (TSK-2000, Bio-Rad) in buffer A (Fig. IA) and then rechromatographed in 0.1 M potassium phosphate buffer in a DEAE anion exchange column. The protein was eluted with a 0 to 200 mM NaCl gradient (Fig. IB). The estimation of the molecular weights of y As was made from the relative mobility of each of the SDS gels (see Example 2 and Fig. 2) and a calibrated gel filtration column. The concordance of the determination of weight between the native forms of the column and the denatured forms in gels indicates that protein A exists in vivo as a monomer. The purification of As as described above results in preparations of more than 95% purity. It has been shown that no protein contaminant from the purified As preparations interferes with the guanosine or adenosine nucleotides in any of the conditions studied. Since the extractions are sequential, the A ,, is purified to obtain an essential homogeneity by the procedure described without detectable contaminants, as shown in the SDS gel described below in Example 2 and represented in Fig. 2, band 1. Once purified, the stability of proteins A differs markedly in aqueous solution. A, is metastable in a purified state and retains most of its functional properties for several days at 4 ° C. I can also withstand freezing and thawing in detergent without losing more than 15 to 20% of its original activity. In contrast, As is labile under a variety of conditions and no satisfactory method of treatment has been found to avoid a loss of activity of more than 80% over a 48 hour period at 4 ° C. The soluble protein A is markedly thermo- and cryolabile. Purified As loses activity rapidly at room temperature (half-life = 2 h) and freezing results in a loss of virtually all activity, presumably due to denaturation and / or aggregation. The purified soluble form is rapidly added in the absence of detergent treatment and is precipitated overnight in the refrigerator under these conditions. EXAMPLE 2 Gel electrophoresis of soluble protein A (AJ and membrane bound (AJ) Polyacrylamide gel electrophoresis was carried out according to a modification of the O 'Farrel methods (J. Biol. Chem., 250: 4001 (1975 )) in the presence of 0.1% SDS in a pore gradient gel (10 to 20%) .The samples were applied in a buffer of samples of 0.33 M DTT, 7% SDS, 17% glycerol and Tris- 0.5 M HCl, pH 6.8, and were operated on until equilibrium, the samples were not denatured by heat, in order to avoid the appearance of additional bands caused by the formation of stable polymers. Bio-Rad silver staining kit The gels were calibrated using Bio-Rad pre-stained molecular weight standards (range 17 to 94 kD) As shown in Fig. 2, "protein A" includes i, a membrane-bound form having a molecular weight of about 20 kD, and As, a soluble form having a mol weight ecular of approximately 19 kD when the gels are worked in reducing conditions. The soluble protein is resolved in two closely spaced bands on the gels. The membrane-bound form migrates as a single band. EXAMPLE 3 Production of murine polyclonal and monoclonal antibodies for protein A Balb / c mice (6-8 weeks of age, The Jackson Laboratory, Bar Harbor, ME) were immunized with four injections of protein A. Injections were performed with a week apart and 50 μg of protein A (either As, or A, or both) was injected on each occasion. The first three injections were administered intraperitoneally and the fourth intravenously. Protein A was injected with complete Freund's adjuvant on the first occasion, incomplete adjuvant on the second and third occasions and without adjuvant on the last occasion. Serum removed prior to the last injection showed a prominent binding of purified protein A using a solid-phase microtiter plate enzyme-linked immunoassay. The mouse was sacrificed with the best immune response three days after the last injection. The lymphocytes of this animal were mained as a polyclonal hybridoma by subcloducing them as antibody producing cells in a Celic bioreactor. These producer cells were kept in cryo-storage in liquid nitrogen. The murine polyclonal antibodies of these producer cells exhibited specificity for protein A. Monoclonal hybridomas were produced by fusion of splenic cells from the sacrificed mouse with NS-1 myeloma cells (P3NS-1/1-Ag4-1) (American Type Culture Collection , Rockville, MD; Access No. TIB18). The method of Nadakavukaren (Differentiation, 27: 209-212 (1984)) was used to carry out the mergers. The resulting clones were studied for protein A binding. Subcloning by serial dilution was carried out in one clone. The most productive subclones were injected into the peritoneal cavity of Balb / c mice to produce ascitic fluid containing monoclonal antibody. The ascitic fluid that was obtained was centrifuged, studied in terms of activity and then stored at -70 ° C until needed. The resulting 18 anti-protein A antibodies were screened selectively for antibody isotype by the Ouchterlony double-diffusion assay on agar plates against anti-IgM, anti-IgG, anti-IgG 1, anti-IgG2a, anti- IgG2b and anti-IgG3 (Cappell). The results are shown in TABLE 1 TABLE 1 EXAMPLE 4 Assays for the binding of GTP and ATP and hydrolysis by protein A, amount of protein and presence of protein A A. GTPase and ATPase assays The rate of hydrolysis of GTP was studied.

ATP by both soluble protein A and membrane bound in a total volume of 200 μl. 1.0 to 10.0 μg of As oir GTP or 5 × 10 ~ 5 M ATP, [? -32P] GTP (2.8 Ci / mmol) 67 to 335 nM or [? -32P] ATP (2 , 8 Ci / mmol) 88.8 to 177.5 nM and 20 μl of purified ROS membranes (in the case of AJ in buffer J (20 mM Tris-HCl, pH 7.0, 0.1 mM EDTA). purified ROS membranes (containing rhodopsin as a receptor) were prepared by washing the ROS membranes 3 times in buffer C (100 mM NaCl, 20 mM Tris-HCl, pH 7.0, 1 mM MgCl 2) and 3 times in water containing polyoxyethylene 23 0.01% lauryl ether (Brij 35 non-ionic detergent, Sigma Chemical Co.) The purified membranes were resuspended in buffer D (10 mM Tris-HCl, pH 7.0, 0.1 mM EGTA) before their The effect of light on the hydrolysis of GTP or ATP was investigated by means of duplicate incubations in light exposure tests (a bright xenon flash of 10 msec (Nikon) that released 1.8 x 103 μWcm ^ sec "1, which was sufficient to whiten more than 70% of the rhodopsin present in each sample) or kept in darkness as controls. The samples were incubated at room temperature for 5 minutes and stopped with 200 μl cold stop buffer at ice temperature (50 nM KH2P04, 6% Norite, TCA 10%). The samples were kept on ice for 30 minutes and rotated for 5 minutes in a microcentrifuge. Aliquots of 50 μl of each supernatant were placed in a vial with 5 ml of scintillation fluid and the radioactivity was studied. The hydrolysis of [? -32P] GTP was measured in the presence and absence of photolized rhodopsin. The GTPase activity of i was increased in the presence of the activated receptor. In contrast, no effect on hydrolysis was observed when un-photolized rhodopsin was added to the incubation in darkness. In the absence of rhodopsin or in the presence of unbleached rhodopsin, i had negligible GTPase activity. In a mol / mol base, the hydrolysis rate of GTP per i (0.458 GTP sec_1 / AJ is comparable to that of transducin (0.512 GTP seg'VTa) when both are measured at submaximal velocity in the presence of photoactivated rhodopsin. GTPase for A, and transducin are additive when the two purified proteins are present in approximately equimolar concentrations Under all the experimental conditions studied, the rate of hydrolysis of GTP (GTPase activity) by the purified As was not sensitive to the presence of rhodopsin bleached or unbleached The apparent constant of Michaelis was determined for As and by measuring the rate of hydrolysis of GTP over a thousand-fold range of substrate concentrations, K ,. and Vmax were determined by construction of double reciprocal plots and regression analysis The data are presented as mean values, and the results are given in TABLE 2.

In the presence of rhodopsin, the K values. for A ,, and As are similar, indicating similar affinities for the GTP. It was found that A, and A, had ATPase activity not associated with receptors. Table 2 above gives the values of K ,. both for the ATPases i and As. The comparison of the rate constants of i indicates that their affinity for the ATP is approximately one order of magnitude greater than that of As. The relative values of K ,. for GTPase and ATPase activity of both A, and As indicate that GTP is the preferred substrate for binding and hydrolysis. The addition of rhodopsin to the incubations did not increase the rate of ATP hydrolysis of any protein. The ATPase rate declines slightly in the presence of the activated receptor. B. GTP binding assays The binding assays of the GTP analogs Gpp (NH) p and GTPγS (New England Nuclear) both by A, as per As were carried out according to the methods of Northup et al. (J. Biol. Chem., 257: 11416-11423 (1982)). The binding was performed in a total volume of 100 μl of solution containing 5 to 10 μg of purified protein A, 3H-Gpp (NH) p (10 μCi) 15.3 μM or GTPγS35 (1 μCi) 1, 32 μM and buffer (100 mM NaCl, 0.1 mM EDTA, 20 mM Tris-HCl, pH 7.0). Samples were vortexed and incubated at 25 ° C for 30 minutes, stopped with 200 μl of buffer cooled to ice temperature (0.5 M NaCl, 0.1 M Tris-HCl, 0.1% Tween 80) and kept on ice for 30 minutes. The samples were placed on nitrocellulose filters that had previously been washed with 2 ml of this same buffer. The filters were washed 5 times with 2 ml of buffer cooled to ice temperature and studied for radioactivity. A ,, bound both GTP? S and Gpp (NH) p spontaneously during a brief incubation at room temperature. In the following TABLE 3 the results are shown. TABLE 3 This procedure apparently did not require cofactors. A, was bound to lower amounts of the este-quiométricas of each GTP analog under the experimental conditions used (TABLE 3). Ace bound to significantly less of these analogs in a mol / mol base after a similar incubation in the absence of cofactors. GTP? S was more easily bound than Gpp (NH) p by both A, and As. C. Competition ATP / GTP The effect of adenosine nucleotides on the binding of Gpp (NH) to a mixture of A was examined. and Ace due to the ability of proteins A to bind ATP and hydrolyze it. Purified i and As (1: 2) were mixed, pre-incubated with ATP or ADP and studied for binding to Gpp (NH) p after a brief incubation by the rapid filtration method. As shown in Fig. 3, ATP was an effective inhibitor of binding at all concentrations studied. ADP was inhibitory in a concentration-dependent manner at higher concentrations. Contrary to the effect of adenosine nucleotides on the binding of Gpp (NH) p, they had negligible effects on the hydrolysis of GTP by protein A. However, at micromolar concentrations, it was found that GTP? S significantly inhibited the hydrolysis of ATP during the course of one hour of incubation (Fig. 4). GTP has a similar, though less pronounced, effect on the hydrolysis of ATP, indicating that both nucleotides compete for the same binding sites or for closely related binding sites on protein A. These results also support the discovery that GTP is a more effective competitor than ATP for protein binding AD Protein assays Protein concentrations were determined according to Bradford (Anal. Biochem., 72: 248 (1976)), using the Bio-Rad Microassay (Bright Blue of Coomassie G-250). Bovine serum albumin was used as standard.

Based on the protein assays performed on the purified species, the complement of i in relation to As (Am / As) is 0.47. The ratio of the amounts of the separate soluble species recovered from high pressure columns (as estimated by optical density at 280 nm) and from plaque gels (as estimated by densitometry) is approximately unity. This indicates that all forms of protein A are extracted in equivalent amounts of rods if the two soluble forms are the products of independent genes. If not, the membrane-bound form would therefore be present at half the concentration of the soluble. E. Assay for protein A The antibodies of this invention produced against protein A were used to create an assay for the detection of that antigen in the serum of humans. The procedure employs an enzyme-linked immunosorbent assay (ELISA). Next, a specific version of this test is presented in Example 8. The type of ELISA used in this context was of the "sandwich" variety. This assay requires two different antibodies that are specific for protein A, where each antibody recognizes and binds to different epitopes on the protein. One of the antibodies serves as a "capture" agent and was used unmodified to coat the bottom of a chamber in a standard 96-well microtiter plate. The unused portion of this antibody was then removed from the well and then a blocking agent (bovine serum albumin (BSA) 1%) was placed in the chamber to block the non-specific binding sites. The purpose serum was then incubated in the well prepared for 1 to 12 hours and then removed. The well was washed with a detergent solution and the second antibody was added in solution to the well. The second antibody used in this procedure is physically bound to a marker substance such as an enzyme; for example, the enzyme could be horseradish peroxidase (HRP). After this incubation, the second antibody was removed and the well was washed once more. The final step was to add an appropriate colorimetric substrate. The specificities of these antibodies for different epitopes in protein A were predetermined by carrying out the ELISA using various combinations of capture enzymes and conjugated with purified antigen (protein A) to determine the possible overlap of the recognition sites. The amount of color development was directly proportional to the amount of enzyme bound antibody bound in the well, which is proportional to the amount of antigen bound in the well by the "capture" antibody. The results were quantified by a spectrophotometric determination of the amount of color produced over a predetermined period of time (typically 30 to 120 minutes). The ELISA described above has been used to study human serum from normal healthy volunteers and from cancer patients for the presence of protein A antigen. The normal population was used to determine the positivity threshold in this assay. The sensitivity of the assay extends to at least the simple ng / ml range as determined by the construction of standard sensitivity curves using known amounts of purified antigen. The population of patients studied in the preliminary use of this trial gave the following qualitative results. A positive reaction was obtained in the trial of patients diagnosed with lung, lymphoma, stomach, colon, rectal and breast cancer compared to normal subjects. EXAMPLE 5 Preparation of rabbit polyclonal antibodies that are immunoreactive with the carboxyl-terminal region of protein A The following procedures were used to generate rabbit polyclonal antibodies that were immunoreactive with the carboxyl-terminal portion of protein A. In one procedure, the carboxyl end of 14 amino acids of the published sequence, QFEPQKVKEKMKNA (SEQ ID NO: 3) of the human protein A was synthesized. A mixture of this synthetic peptide was injected subcutaneously in Freund's adjuvant at 3-4 separate sites in each rabbit. The amount of peptide synthesized in each injection was 50 μg. At two weeks, booster injections of 50 μg of peptide synthesized in Freund's adjuvant were given in 3-4 separate sites in each of the previously inoculated animals. After two weeks, test bleeds were made to titrate the antibody titers in blood with the synthesized peptide. More booster injections were made as necessary to maintain adequate titers of antibodies in blood. Sera from rabbits with suitable antibody titers in blood were collected and the desired antibodies were obtained as a purified fraction by affinity chromatography techniques using the immobilized carboxy terminus peptide immobilized on a solid matrix. The purified antibodies were precipitated by dialysis in water and stored as dry powder for later use. These polyclonal rabbit antibodies were reactive with the peptide derived from the carboxyl-terminal region of protein A and were designated CY2A antibodies. In another procedure, a peptide sequence of approximately 16 amino acids was produced synthetically at the carboxyl terminus of protein A. This peptide was then conjugated to the potent immunostimulatory molecule (adjuvant): limpet hemocyanin (KLH). The peptide-KLH conjugate was then injected into rabbits by standard procedures used to generate antibodies. The rabbits were subsequently bled and the sera were studied and showed to be positive for the carboxyl-end peptide of protein A used in the immunogen. These polyclonal rabbit antibodies were termed CPDD antibodies.

EXAMPLE 6 Assay for the presence of protein A in a liquid sample In order to detect the presence of protein A in a specified sample, one of the following procedures was carried out: A. Aliquots of a standard solution containing one of the monoclonal antibodies of Example 3 (antibodies 3B9E1) in each well of a 96-well titration tray and allowed to dry so that the residual antibodies adhered to the substrate at the bottom of each well. Aliquots of 1% bovine serum albumin solution were added to each well for blocking purposes. The residual liquid was separated. From each test sample, 50-100 μl of the sample was placed in specified wells and incubated for one hour at room temperature. The liquid portion was decanted and each well was washed with phosphate buffered saline (PBS). Aliquots of a standard solution containing the antibodies of Example 5 (CY2A antibodies), to which horseradish peroxidase had been attached, were applied to each well and incubated for one hour at room temperature. The liquid portions were removed and each well was washed with PBS. Finally, aliquots of the horseradish peroxidase substrate, diammonium salt of 2,2'-azinodi [3-ethylbenzthiazoline sulfonate (6)] (ABTS) were added and the development was allowed to take place for one hour at 37 ° C. . The degree of color development for each well was quantified obtaining a spectrophotometric reading at 410 nm. These color measurements were converted to units by comparison with a calibration curve.

B. In another procedure, a biotin / streptavidin detection system was used. The CPDD antibody of Example 5 was first conjugated to biotin using covalent binding chemistry. An activated N-hydroxy-succinimide ester of biotin was allowed to react with purified antibody, such that covalent attachment of the biotin molecules to primary amines on the antibody molecule was produced. The unreacted biotin was removed by chromatography. The antibody was then titrated with the immunizing antigen to find the optimal assay dilution. In general, the procedure was carried out as follows: The biotinylated antibody was first allowed to bind to its target antigen. Simultaneously, a second antibody immobilized on a solid phase support (microtiter plate) will capture the same antigen. After removing any unbound antibody by washing, the antibody complex was reacted: antigen: biotinylated antibody with streptavidin conjugated to a reporter molecule, typically an enzyme such as horseradish peroxidase. Another washing step was performed to eliminate streptavidin: unbound enzyme. It was then left that a specific substrate for the enzymatically labeled streptavidin will react with the rest of the streptavidin: enzyme complex. The amount of substrate hydrolyzed to the chromogenic product was thus directly proportional to the amount of antigen present in the sample. The advantage of the biotin / is-treptavidin detection system is that the antibody can be gently labeled with multiple biotin molecules without loss of antibody activity. The fact that more than one biotin is present in each antibody molecule allows the signal to be amplified through the subsequent binding of multiple streptavidin molecules. Specifically, the procedure employing the biotinylated antibodies was carried out in the following manner: The aliquots of a standard solution containing one of the monoclonal antibodies of Example 3 (the 3B9E1 antibodies) were placed in the individual wells of a microtiter plate . The antibodies were allowed to adsorb on the bottom and sides of each well. The residual solutions were aspirated from the wells and aliquots of a 1% bovine serum albumin solution containing 20% sucrose were added to each well for blocking purposes. The residual liquid was removed.

The subsequent reagents were initially allowed to warm to room temperature. The patient samples (plasma with EDTA) were mixed and, if any particulate matter was observed, the sample was clarified by centrifugation. An aliquot of 50 μl of patient plasma and 200 μl of biotinylated rabbit polyclonal CPDD antibody in 0.05 M phosphate buffered saline at pH 7.4 were mixed with 1 mg / ml bovine serum albumin and added to the wells. individual These solutions were allowed to incubate in the wells for 2 hours. They were then aspirated and the wells were washed 3 times with aspiration. At this point in the procedure, they were added 200 μl of streptavidin enzyme conjugate: horseradish peroxidase to each well and allowed to incubate for 1 hour at room temperature. The wells were then washed and aspirated 3 times as before. After this step, 200 μl of tetramethylbenzidine substrate was pipetted into each well and incubated for 20 minutes at room temperature. Then 100 μl of stop solution (0.05 N sulfuric acid) was added. The absorbance of each well was read on a spectrophotometer of double wavelength microtitre plates at 450/630 nm. The mean absorbance for each sample was computed. (If the duplicates differed by more than 10%, the sample was repeated). Samples with off-scale absorbance readings were diluted and re-studied. The mean of each sample was divided by the mean of the negative controls to give a P / N value. Samples with a P / N greater than 2.0 were given as positive. To establish the baseline for the value of the negative control (N), 40 negative samples were initially studied in duplicate to identify those that were most appropriate to establish a cut between positive and negative samples. A geometric mean was then calculated by eliminating the two samples with higher and lower absorbance and re-calculating the mean and the standard deviation of the remaining samples. The cut for positivity was then arbitrarily set at 2 standard deviations above the mean for the remaining samples. At the same time, the mean plus 2 standard deviations was very close to 2 times the average. Hence, some trials used a positivity cutoff of 2.0 when the unknown sample was divided by the mean of the negative control samples to establish a so-called P / N ratio. Next, a negative human serum conjugate serving as the test / standard control was obtained. This material had a mean absorbance essentially identical to the geometric mean of the previous negative samples. Therefore, this negative control was calibrated against negative sera to serve as control of the assay. This test control set was worked in duplicate for each test and the mean absorbance was calculated with the positivity cut stipulated as 2 times the mean of the negative control. EXAMPLE 7 Selective study procedure to predict the presence of metastatic cancer Blood samples were collected from several individuals. The group of individuals included people diagnosed with metastatic breast cancer, people from a control population without signs of cancer and people who had been previously diagnosed as having had cancer, but who, at the time of taking blood samples, They were diagnosed with breast cancer in a state of remission. A portion of the blood sample from each individual was used as a sample in the test of Example 6A. The test results for the blood sample of each individual appear in Table 4.

TABLE 4 The diagnosis marked as SEE refers to no evidence of recurrent disease. The results of this trial also appear in Figure 1. The mean result of the trial for the combination of individuals identified as controls and people whose cancer was in a remission state is 4., 5 ng of protein A / ml of blood. The mean result of the trial for individuals with metastatic breast cancer is 23.4 ng of protein A / ml of blood. With the exception of an individual who was diagnosed as having metastatic cancer, but had a test result within the range of controls and SEE, all trial results could be clearly divided into two distinct groups: those individuals who had no signs of cancer and those individuals who had metastatic breast cancer. This delimitation is not usual. The statistical significance of these results was p <; 0.001 for a two-tailed t test. EXAMPLE 8 Selective Study Procedure for Predicting the Presence of Primary as Well as Metastatic Cancer In an extensive selective study procedure, blood plasma samples from more than eight hundred human patients were subjected to the test protocol of Example 6B. The patient population included individuals without cancer (controls), individuals with various stages of primary cancers, individuals with metastatic cancer and individuals with benign tumors. The test procedure was carried out in a blinded manner and the protein A assay results were subsequently aligned with independent clinical diagnoses for trial verification purposes. The results of this test procedure are shown in Table 5. The type of cancer, the stage of the cancer and the number of individuals correctly assessed by the test is compared to the results of an independent diagnosis (sensitivity).

TABLE 5 Type of cancer Positive in the trial / # total samples Mama 24/35 = 69% Stage 1 1/6 2 3/4 4 1/2 19/23 Primary liver 17/25 = 68% Stage 2 1/1 3 5/7 3b 1/1 4 2/4 4a 7/9 4b 1/3 Pancreas 6/9 = = 67% Stage 4 6/8 4a 0/1 Prostate 24/41 = 61% Stage D2 0/2 25/39 Bladder 11/19 = 58% Lymphoma 18/32 = 56% Stage? 2/3 1 0/1 1/1 lb 0/1 2nd 1/4 2B 0/1 2bc 1/1 2c 1/1 2nd 1/1 3b 3/4 4 3/6 4th 0/1 4B 4 / 4 TABLE 5 (continued) Type of cancer Positive in the trial / # total of samples 4b 1/3 Head and neck 9/16 = 56% Stage 3 1/2 4 4/10 4/4 Lung 41/91 = = 45% Stage 1 4/5 2 2/4 3a 2/5 3b 6/13 4 10/27 ED 4/6 LD 2/7? 11/24 Colon and rectum 17/43 = 40% Stage 2 0/1 4 1/3 Bl 2/2 B2 1/4 C2 2/4 D 3/3 8/26 Leukemia 10/26 = 38% Multiple myeloma 5/5 = 100% Stage 3a 3/3 3b 2/2 Endometrium 5/5 = 100% Stage 4 1/1? 4/4 Parotid 4/4 = 100% Colangio CA 3/10 = 30% Stage 3 1/1 4 2/8 TABLE 5 (continued) Type of cancer Positive in the trial / # total of samples 4a 0/1 Kidney 2/6 = 33% Stage 3 1/2 4 1/2? 0/2 Cervix 3/4 = 75% Stage 4 1/1 4th 1/1? 1/2 Thyroid 2/3 = 67% Stage 1 1/1 4 1/2 Brain 4/6 = 67% Mouth 2/3 = 67% Uterus 2/7 = 29% Metastasis 2/7 = 29% (unknown origin) Melanoma 2/2 = 100% Stage 4 2/2 Ovary 0/2 = 0% Abdominal 1/2 = 50% Urinary 0/2 = 0% Tongue 1/3 = 33% Lip 1/1 = 100% Anal 1/1 = 100% Pelvic 1/1 = 100% Inguinal 1/1 = 100% Penis 1/1 = 100% Rib 1/1 = 100% Horn 1/1 = 100% Fallopian Sarcomas 2/7 = 29% POEMS 1/1 = 100% Laringe 0/2 = 0 % TABLE 5 (continued) Type of cancer Positive in the trial / # total of samples Cells 0/1 = 0% germinal Spinal cord 0/1 = 0% Testicular 0/1 = 0% Vulva 0/1 = 0% Spleen 0/1 = 0% Summary of all cancers 262/518 = 51% In addition, the test was performed for normal individuals ( non-cancerous state) and for people with benign tumors. The results are shown in Table 6, where a "correct" test result (proper determination that the individual does not have cancer) occurred when the P / N ratio for that individual was less than or equal to 2.0.

TABLE 6 The results of these trials show that the test procedure has a marked predictive capacity to discern individuals with cancer, while eliminating those individuals who do not have cancer. Cancers detected by this assay include primary as well as metastatic cancers. Cancer is detected in several stages, including stage 1 and stage 2. Specifically, breast cancer, prostate cancer, primary liver cancer, lymphoma, pancreatic cancer, lung cancer, cancer colon, bladder cancer, endometriotic cancer and multiple myeloma are easily detectable with the present trial. Equivalents Having particularly shown and described this invention in relation to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined. by the appended claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Schepens Eye Research Institute (B) STREET: 20 Staniford Street (C) CITY: Boston (D) STATE / PROVINCE: Massachusetts (E) COUNTRY: USA (F) ZIP / ZIP CODE: 02114 (G) TELEPHONE: 617-742-3410 (I) TELEFAX: 617-720-1069 (i) APPLICANT: (A) NAME: CYTRA CORPORATION (B) STREET: 82 Beachside Avenue ( C) CITY: Greens Farms (D) STATE / PROVINCE: Connecticut (E) COUNTRY: USA (F) ZIP / ZIP CODE: 06436 (G) TELEPHONE: 203-259-6876 (I) TELEFAX: 203-255-2386 (i) APPLICANT / INVENTOR: (A) NAME: Geoffrey J. Schmidt (B) STREET: 73 Tiffany Road (C) CITY: Norwell (D) STATE / PROVINCE: Massachusetts (E) COUNTRY: USA (F) POSTAL CODE / ZIP: 02061 (i) APPLICANT / INVENTOR: 5 (A) NAME: Kenneth L. Hoffman (B) STREET: 95 Comstock Drive (C) CITY: Wrentham (D) STATE / PROVINCE: Massachusetts (E) ) COUNTRY: USA 10 (F) POSTAL CODE / ZIP: 02093 (ii) TITLE OF THE INVENTION: PROTEIN A AS DIAGNOSIS OF CANCER (iii) NUMBER OF SEQUENCES: 3 (iv) ADDRESS FOR CORRESPONDENCE: 15 (A) RECIPIENT: Hamilton, Brook, Smith &; Reynolds, P.C. (B) STREET: Two Militia Drive (C) CITY: Lexington (D) STATE: Massachusetts 20 (E) COUNTRY: USA (F) ZIP: 02173 (v) COMPUTER FORM OF THE COMPUTER: (A) TYPE OF MEDIUM: Floating disk (B) COMPUTER: IBM Pe Compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM: Patentln Reread # 1.0, Version # 1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) APPLICATION DATE: (C) CLASSIFICATION: (vii) DATA FROM PREVIOUS APPLICATION: (A) APPLICATION NUMBER : US 08 / 370.969 '(B) APPLICATION DATE: 10-JANUARY-1995 (viii) INFORMATION ON THE POWDER / AGENT: (A) NAME: Wagner, Richard W. (B) REGISTRATION NUMBER: 34,480 (C) NUMBER REFERENCE / RECORD: SERI88- 01A3 PCT (ix) TELECOMMUNICATIONS INFORMATION: (A) PHONE: (617) 861-6240 (B) TELEFAX: (617) 861-9540 (2) INFORMATION FOR SEQ ID NO: 1 : (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1 : Gly Asn Ser Lys Ser Gly Ala Leu Ser Lys Glu lie 1 5 10 Leu Glu Glu Leu Gln 15 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 2: Met Gly Asn Ser Lys Ser Gly Ala Leu Ser Lys Glu 1 5 10 lie Leu Glu Glu Leu Gln 15 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: Gln Phe Glu Pro Gln Lys Val Lys Glu Lys Met Lys 10 Asn Ala

Claims (36)

1. CLAIMS 1. A method for detecting the presence of cancer in an individual, consisting of: (a) obtaining a biological sample of said individual; (b) incubating said biological sample with at least one antibody immunoreactive with protein A; (c) detecting the immunoconjugates that are formed as a consequence of the incubation of step (b), and (d) relating the amount of immunoconjugates of step (c) to the presence of cancer, where the cancer is present when said amount is greater than a threshold value.
2. 2. A method of detecting the presence of a cancerous state in an individual, consisting of: (a) obtaining a liquid sample from said individual, (b) submitting said liquid sample to an antibody sandwich assay, wherein one of the two antibodies is immunoreactive with a peptide having a specified sequence of about 16 amino acids of the amino acid sequence of protein A and the other antibody is immunoreactive with a portion of protein A other than said peptide with the specified protein A sequence of about 16 amino acids, (c) detecting the amount of antibody-protein sandwich A formed in step (b) and (d) relating said amount to the presence of a cancerous state in said individual, wherein said cancerous state is present when said quantity is greater than a threshold value.
3. 3. The method of Claim 2, wherein at least one of said two antibodies is a monoclonal antibody.
4. The method of Claim 3, wherein the antibody that is immunoreactive with a portion of the protein A other than said peptide with the specified protein A sequence of about 16 amino acids is a monoclonal antibody.
5. The method of Claim 4, wherein the antibody that is immunoreactive with a peptide having a specified sequence of about 16 amino acids of the amino acid sequence of protein A is a polyclonal antibody with a linked detection label.
6. 6. The method of Claim 2, wherein the liquid of said liquid sample is blood.
7. The method of Claim 6, wherein: (i) the antibody that is immunoreactive with a portion of protein A other than said peptide with the specified protein A sequence of about 16 amino acids is a monoclonal antibody adherent to a solid surface and (ii) the antibody that is immunoreactive with a peptide having a specified sequence of about 16 amino acids of the amino acid sequence of protein A is a polyclonal antibody with a bound detection label.
8. 8. The method of Claim 7, wherein said bound detection label is biotin.
9. The method of Claim 7, wherein said threshold value is 2.0 times the average of the value of the negative control.
10. The method of Claim 9, wherein said cancerous state is selected from the group consisting of breast cancer, prostate cancer, primary liver cancer, lymphoma, pancreatic cancer, lung cancer, colon cancer, bladder cancer, endometrial cancer and multiple myeloma.
11. The method of Claim 7, wherein said specified sequence of about 16 amino acids of the amino acid sequence of protein A is either amino acids 142-158 of protein A, or the carboxyl terminus of 16 amino acids of the protein A.
12. 12. A method of detecting a cancer diagnostic protein, whose elevated level in the bloodstream of an individual predicts that the individual has cancer, consisting of: (a) incubating a liquid sample of said individual with an antibody that immunoreacts with a peptide having a specified sequence of about 16 amino acids of the amino acid sequence of said cancer diagnostic protein and with an antibody that immunoreacts with a portion of said cancer diagnostic protein other than said peptide with the sequence of the diagnostic protein of the cancer. specified cancer of approximately 16 amino acids, whereby immunocomplexes are formed between said cancer diagnostic protein and the two antibodies, and (b) detecting the amount of said immunocomplexes formed in step (a).
13. The method of Claim 12, wherein the liquid of said liquid sample is blood.
14. 14. The method of Claim 13, wherein said liquid sample is incubated simultaneously with the two named antibodies.
15. The method of Claim 14, wherein: (i) said antibody that immunoreacts with a peptide having a specified sequence of about 16 amino acids of the amino acid sequence of said cancer diagnostic protein is a polyclonal antibody with a detection label bound and (ii) said antibody that immunoreacts with a portion of said cancer diagnostic protein other than said peptide with the specified cancer diagnostic protein sequence of about 16 amino acids is a monoclonal antibody adherent to a solid surface.
16. 16. The method of Claim 15, wherein said bound detection label is biotin.
17. 17. The method of Claim 13, wherein said liquid sample is incubated simultaneously with the two said antibodies.
18. The method of Claim 13, wherein said liquid sample is first incubated with said antibody that immunoreacts with a portion of said cancer diagnostic protein other than said peptide with the specified cancer diagnostic protein sequence of about 16 amino acids and then incubated with said antibody that immunoreacts with a peptide having a specified sequence of about 16 amino acids of the amino acid sequence of said cancer diagnostic protein.
19. The method of Claim 18, wherein: (i) said antibody that immunoreacts with a portion of said cancer diagnostic protein other than said peptide with the specified cancer diagnostic protein sequence of about 16 amino acids is an adherent monoclonal antibody to a solid surface and (ii) said antibody that immunoreacts with a peptide having a specified sequence of about 16 amino acids of the amino acid sequence of said cancer diagnostic protein is a polyclonal antibody with a bound detection label.
20. 20. The method of Claim 15, wherein said cancer diagnostic protein is protein A.
21. 21. The method of Claim 20, wherein an amount of said immunocomplex greater than 2.0 times the mean value of the negative control is predictive of that said individual has cancer.
22. 22. The method of Claim 20, wherein said specified sequence of about 16 amino acids of the amino acid sequence of said cancer diagnostic protein is either amino acids 142-158 of said cancer diagnostic protein or the carboxyl terminus of 16 amino acids. of said cancer diagnostic protein.
23. 23. A method of detecting the presence of metastatic cancer in an individual, consisting of: (a) obtaining a biological sample from said individual, (b) incubating said biological sample with at least one antibody that is immunoreactive with protein A, ( c) detecting immunoconjugates that are formed as a consequence of the incubation of step (b), wherein an abnormally large amount of said immunoconjugates detected in step c) is indicative of the presence of metastatic cancer in said individual.
24. 24. The method of Claim 23, wherein said biological sample is blood.
25. 25. The method of Claim 24, wherein the cancer is breast cancer.
26. 26. The method of Claim 23, wherein one antibody is the EB9E1 antibody and another antibody is CY2a.
27. 27. An antibody that binds to protein A to form immunoconjugates, whose presence in an abnormally large amount in a biological sample is predictive of the presence of cancer in the individual from which said biological sample was obtained.
28. 28. The antibody of Claim 27, where said biological sample is blood.
29. 29. The antibody of Claim 28, wherein said antibody is selected from the group consisting of the 3B9E1 antibody, the CY2a antibody and the CPDD antibody.
30. 30. A test kit for detecting the presence of cancer in an individual, consisting of: (a) a first antibody that immunoreacts with a peptide having a specified sequence of about 16 amino acids of the amino acid sequence of a cancer diagnostic protein and (b) a second antibody that immunoreacts with a portion of said cancer diagnostic protein other than said peptide with the specified cancer diagnostic protein sequence of about 16 amino acids.
31. 31. The assay kit of Claim 30, wherein either said first antibody or said second antibody has an identification tag attached.
32. 32. The assay kit of Claim 30, wherein at least one of said first antibody and said second antibody is a monoclonal antibody.
33. 33. The assay kit of Claim 32, wherein said first antibody is a polyclonal antibody with a binding identification label and said second antibody is a monoclonal antibody adherent to a solid surface.
34. 34. The test kit of Claim 33, wherein said attached identification label is biotin.
35. 35. The test kit of Claim 30, wherein said cancer diagnostic protein is protein A.
36. 36. The assay kit of claim 35, wherein said specified sequence of about 16 amino acids of the amino acid sequence of said diagnostic protein of the cancer is either amino acids 142-158 of said diagnostic cancer protein or the carboxyl-terminal 16 amino acids of said diagnostic cancer protein.