MXPA99008774A - Method for determining the presence of mutated brca protein - Google Patents
Method for determining the presence of mutated brca proteinInfo
- Publication number
- MXPA99008774A MXPA99008774A MXPA/A/1999/008774A MX9908774A MXPA99008774A MX PA99008774 A MXPA99008774 A MX PA99008774A MX 9908774 A MX9908774 A MX 9908774A MX PA99008774 A MXPA99008774 A MX PA99008774A
- Authority
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- Mexico
- Prior art keywords
- protein
- brca
- cells
- antibody
- mutated
- Prior art date
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Abstract
This invention relates to a simple and reliable screening method for determining an individual's susceptibility to breast, ovarian, colon or prostate cancer. This invention also relates to a test kit for use in conjunction with this method. The inventive method targets a specific alteration of the BRCA protein and identifies a non-invasive source of normal cells that express this protein.
Description
METHOD FOR DETERMINING THE PRESENCE OF BRCA MUTADA PROTEIN
FIELD OF THE INVENTION
The present invention relates, in general, to a method for determining the susceptibility of an individual to cancer of the breast, ovaries, colon or prostate.
BACKGROUND OF THE INVENTION
The genetic mutations of BRCA-1 and BRCA-2 have been identified as responsible for approximately 90% of all early-onset, hereditary breast and ovarian cancers. The genetic mutations of BRCA-1 and BRCA-2 have also been associated with an increased risk, or susceptibility, to colon or prostate cancer. The complete sequence of the BRCA-1 and BRCA-2 genes was discovered and / or published in October 1994 and December 1995, respectively. The availability of these sequences and the amino acid sequences
^ F .: 85215
deduced has allowed the generation of representative peptide sequences. These peptide sequences have been used as immunogens that give rise to antibodies capable of detecting specific regions of the BRCA-1 and BRCA-2 proteins. It has been reported that approximately 88% of the BRCA-1 mutations result in the production of a reduced or shortened protein, which has lost the sequences of the terminal ends of the BRCA-1 protein (ie, the carboxy terminus). (term C)). (See Donna Shattuck-Eidens et al., A Co llaborative S rvey of 80 Mutatlons ..., J. A. Med. Ass'n, Vol. 273 No. 7, February 15, 1995, on pages 535- 541). Data are available that document the degree of the BRCA-2 mutations and that also result in truncation of the protein. (See Simon A. Gayther et al., Varlation of Risks of Breast and Ovarian Cancer ... BRCA-2 gene, NATURE GENETICS, Vol. 15, 1997, January 1997, on pages 103-105). However, it is reported that BRCA-1 mutations have taken the form of alterations in a single base, deletions of
a single base or multiple base deletions resulting in trans internal locations of the table, ending with stop codons located downstream (direction of the nucleotide sequence extending beyond the 3 'end of the last exon of a gene), producing the truncation of the BRCA-1 protein consequent to the presence of the mutation of a gene. As is well known to those skilled in the art, a normal individual carries two untreated alleles of the BRCA-1 protein while a susceptible individual inherits a normal allele from a parent and a mutated allele from the mother. These pairs of normal and heterozygous alleles are carried in each nucleated cell within the body. In tumor tissues normal alleles have frequently been altered leaving the susceptible individual without normal genetic copies to produce the normal, full-length BRCA-1 protein. In view of the above, the inventors of the present have developed the hypothesis that normal nucleated cells of an affected individual will express a normal copy of the protein
BRCA-1 and a truncated copy, while it is likely that its tumor cells possess two altered copies of the protein. Antibodies capable of detecting the amino terminus (N-terminus) of the BRCA-1 protein and antibodies capable of detecting the C-terminus of the BRCA-1 protein are known. These antibodies have been used to localize the BRCA-1 protein in particular regions of normal cells and tumor cells (See Ralph Scully et al., Location of BRCA-1 in Human Breast Cells and Ovarian Cells, SCIENCE, Vol. 272, 5 April 1996, pages 123-124) and, more recently, have been used to inspect the expression and localization of proteins expressed from transfected constructs containing selected regions of the BRCA-1 protein (See Cindy A. Wilson et al., Differential Subcellular Localization Expression and Biological
Toxicity ..., ONCOGENE, Vol. 14, January 1997, on pages 1-16). Antibodies capable of detecting the N-terminus and the C-terminus regions of the BRCA-2 protein are also available.
Based on some of the findings referred to above, Myriad Genetics, Inc. of Salt Lake City, Utah, developed a comprehensive DNA sequence analysis of BRCA-1 and BRCA-2, which is marketed under the commercial designation of BRACAnal ys i sMR, extensive analysis of sequences of BRCA-1 and BRCA-2, for susceptibility to breast and ovarian cancer. This genetic analysis reportedly identifies genetic mutations in all sequences encoding the protein and in additional adjacent areas, both in the BRCA-1 and BRCA-2 genes. The cost or rights to carry out this genetic analysis, is said to be $ 2,400 US dollars. Due to the complex and extensive nature of this genetic analysis based on DNA, and the resulting higher cost, it is used primarily for individuals who are at high risk of breast or ovarian cancer, hereditary, and those with a diagnosis of cancer. of breast (especially premenopáus i co) or of ovaries. In addition to the above,
WO 96/33271, (WO'271) describes a delayed and laborious antibody-based method for diagnosing a patient's susceptibility to cancer, which targets specific and uniquely mutated regions of a protein, and which requires a relatively large sample of cells that can not be easily obtained in a non-invasive manner. For the method of WO'271 to accurately diagnose a patient's susceptibility to cancer, resulting from a localized mutation within the BRCA-1 protein, it would be necessary to analyze each region within the BRCA sequence, with a specific antibody. for the region. Similarly, EP-A-0705903 discloses a time-consuming and laborious antibody-based method for detecting specific mutations of the BRCA 1 protein, which requires the use of multiple antibodies to identify discrete regions of the protein. To determine if there is a mutation in the BRCA 1 gene sequence, each region within the BRCA 1 sequence would need to be analyzed, necessitating the generation of
approximately 300 antibodies, to represent the total protein. As mentioned above, this approach requires large samples of cells that can not be obtained quickly or easily from a cell source with a non-invasive procedure. Accordingly, there is a need to develop a less complex and therefore less costly method to determine susceptability to cancer. Therefore, the objective of the present invention is to develop a selective, general classification method to determine the susceptibility to breast and ovarian cancer, and to other types of cancer associated with that susceptibility. A more particular objective is to develop an antibody-based method to detect the reduction or loss of regions of the C-terminus of the BRCA-1 or BRCA-2 protein, which is indicative of a genetic mutation. Another object of the present invention is to develop a test or analysis kit for use in conjunction with the method developed.
BRIEF DESCRIPTION OF THE INVENTION
The present invention therefore provides an antibody-based method for determining the susceptibility of a patient to certain types of cancer, and the method is directed to a specific alteration or mutation, of a protein expressed in epithelial cells considered responsible for the emergence or early development of those cancers, where a clinically significant percentage of these protein mutations result in the production of a reduced or tened protein, from which a specific sequence or region has been lost. The present invention more particularly provides an antibody-based method for determining the presence of the mutated BRCA-1 or BRCA-2 protein (hereinafter referred to, in general, as "BRCA protein") in cells expressing this protein, where the method comprises:
prepare a first and a second equal amount of cells that express the
BRCA protein; administering an amount of a first primary antibody, to the first number of cells, and an equal amount of a second primary antibody, to the second number of cells; measuring the reactivity of the antibodies in the first and second amounts of cells; and comparing the measured reactivities of the antibodies, to determine the presence of mutated BRCA protein, in the first and second number of cells; wherein the unmutated BRCA protein contains the amino and carboxy terminus regions, wherein the mutated BRCA protein is typically a shortened or reduced protein, from which the carboxy terminus regions have been lost, wherein the first primary antibody used in the inventive method is capable of detecting or reacting with the amino-terminus regions of the BRCA protein, wherein the second primary antibody is capable of detecting or reacting with the
regions of the carboxy terminus of the BRCA protein, and wherein a difference in the mediated reactivities of the antibodies, indicates the presence of the mutated BRCA protein, in the first and second number of cells.
The present invention also identifies a non-invasive source of normal cells, which expresses the BRCA protein and provides kits for testing or analysis, for use in conjunction with the antibody-based method, referred to above. The preceding features and advantages, as well as others, of the present invention will become apparent from the following description.
DESCRIPTION OF THE PREFERRED MODALITY
The present inventive method is directed to a specific alteration of the BRCA protein, especially the reduction or loss of the C-terminus regions of these proteins.
Due to the high incidence of BRCA gene mutations, which results in reduced or shortened proteins, from which these regions have disappeared, the present method is considered a simple and reliable selective classification technique that provides a positive and clear result. However, the inventive method could provide an ambiguous negative result. In particular, a positive result in the method or assay described herein can be considered as definitive proof that the individual subjected to the analysis carries a mutated allele. A negative result would indicate, either, that the individual falls within 12% of those individuals whose allelic mutations fail to produce the truncation of the protein, or that the individual carries a non-truncated allele. Through the present invention, a non-invasive source of normal cells expressing the BRCA-1 protein (and to a lesser extent the BRCA-2 protein) has been identified. As will be readily apparent to those skilled in the art, the availability of a non-invasive source of normal cells
expressing these proteins, greatly reduces the cost of the selective classification technique of the invention and allows it to be used in conjunction with special containers for mailing or with test kits that serve as a means for the unaided collection of samples of cells, of individuals that undergo this selective classification analysis. The non-invasive source of normal cells, which has been identified, constitutes epithelial cells of the inner cheeks (ie, buccal cells). These cells can be scraped easily with a cotton swab, deposited by the subject to be analyzed, in a liquid medium, and sent by mail to a center to perform the analysis. As mentioned above, the inventive method herein is based on the hypothesis that the cells of an affected individual express 50% of the BRCA protein having equal numbers of regions with the N term and with the C term, and the 50% of the BRCA protein that has a reduced number of
regions with the term C. This observed imbalance, between the regions of the N term and the C term, in these proteins, is detected by: the administration of an antibody capable of detecting or reacting with the N-terminus regions, for a first sample of cells; the administration of an antibody capable of detecting or reacting with the regions of the term C, for a second equivalent sample of cells; the measurement of the reactivities of the antibodies in the cell samples; and comparing the measured reactivities of the antibodies, to determine the presence of the mutated BRCA protein in the cell samples. Any difference between the measured reactivities of the antibodies would indicate the presence of truncated copies of the protein. The present invention is suitable for use with cells that express the BRCA protein. These cells can be obtained from any region of the human body that is free of tumorous or precancerous tissue, including, but not limited to, blood leukocytes, skin and nodes
lymphatics These cells can also be obtained from tumorous tissue that includes the tumorous tissue of the breast, ovaries, colon and prostate. As mentioned hereinabove, it is preferred that the source of cells used in the present invention constitute a non-invasive source such as epithelial cells, and more preferably, oral cells. The preparation of the cells referred to above, for the joint use with the inventive method hereof, can be carried out according to conventional methods and techniques. For example, freshly isolated single monodi-spher cells can be deposited by centrifugation and then fixed in a 50:50 acetone-methanol solution. The tissue, instantly frozen in liquid nitrogen, can be cut under cryostat, and once fixed in buffered formalin (= 4% formaldehyde) it can be embedded in paraffin and then cut with a microtome. In a preferred embodiment, single cells and small clusters of buccal cells are obtained, vigorously rubbing the
inner cheek of a subject to be subjected to analysis, with a cotton swab moistened with phosphate buffered saline (PBS, for its acronym in English). The cotton swab is then immersed in an amount of PBS contained in a vial and the cells are freed from it by shaking. The cells are then counted (for example, using a hematocrit), diluted to 20,000 cells / ml, deposited by centrifugation, fixed in an acetone-methanol solution, 50:50, and stored at -20. ° C. The cells can be stored at this temperature, in sealed boxes, for a period of 1 to 4 months. In a more preferred embodiment of the present inventive method, the imbalance between the N-terminus and the C-terminus regions of the BRCA protein is detected by: the administration of an amount of an antibody that reacts with the N-terminus regions of the protein (referred to hereinafter as "primary" antibody) for a first sample of cells; the addition of an equal amount of another
antibody that reacts with the regions of the C-terminus of the protein (hereinafter also referred to as the "primary" antibody) for a second equivalent cell sample, the administration of a quantity of a secondary antibody labeled with biotin (which it serves as a color detection device, by reaction with a determinant in the primary antibodies) for the first and second cell samples; the addition of an amount of an enzyme complex (which serves to mark the location of the antibody reactivity within the test sample) for the respective cell samples; and then, the dyeing and counterstaining of the cells in each sample, before the determination and comparison of the reactivity of the primary antibody. Primary antibodies capable of detecting the N-terminus regions of the BRCA-1 protein include the antibodies prepared by immunization using a peptide sequence representative of amino acids 2-21 of the BRCA-1 protein. In a preferred embodiment, an IgG antibody is employed
polyclonal rabbit sold by Santa-Cruz Biotechnology, Inc., 2161 Delaware Avenue, Santa Cruz, CA 95060-5706, under the trade designation "BRCA-1 D-20" (product No. sc-641). Primary antibodies capable of detecting the C-terminus regions of the BRCA-1 protein, which are suitable for use in the present invention, include antibodies prepared by immunization using a peptide sequence representative of amino acids 1843-1862 of the BRCA-protein. 1. In a preferred embodiment, a polyclonal rabbit IgG antibody sold by Santa-Cruz Biotechnology, under the trade designation "BRCA-1 C-20" (product No. sc-642) is employed. Primary antibodies capable of detecting the N-terminus regions of the BRCA-2 protein include antibodies prepared by immunization using a peptide sequence representative of amino acids 3-19 of the BRCA-2 protein. In a preferred embodiment, a goat polyclonal IgG antibody, sold by Santa-Cruz Bí or t etchnolo gy, Inc., is used.
the commercial designation of "BRCA-21-17" (product No. sc-1818). Primary antibodies capable of detecting the C-terminus regions of the BRCA-2 protein, which are suitable for use in the present invention, include antibodies prepared by immunization using a representative peptide sequence of amino acids 3404-3418 of the BRCA protein. -2. In a preferred embodiment, a goat polyclonal IgG antibody sold by Santa-Cruz Biotechnology, Inc. under the trade designation "BRCA-2 C-15" (product No. sc-1816) is employed. These primary antibodies are preferably diluted with PBS to concentrations ranging from about 0.5 micrograms
(μg) / milliliter (ml) of PBS to approximately
1. 0 μg / ml for BRCA-1 or from approximately 2.0 to approximately 5.0 μg / ml for
BRCA-2 and is administered in an amount of 20,000 cells, in amounts ranging from about 0.1 to about 2 ml
(ie, from about 0.05 μg to about 2.0 μg of primary antibody
for BRCA-1 or from approximately 0.2 μg to approximately 10.0 μg of primary antibody for BRCA-2). Secondary antibodies capable of binding to primary antibodies, which are suitable for use in the present invention, include the antiserum obtained from goats immunized with rabbit immunoglobulin, for the detection of primary antibodies to BRCA-1, and the antiserum obtained. of rabbits immunized with goat immunoglobulin, for the detection of primary antibodies for BRCA-2. In a preferred embodiment, an affinity purified anti-immunoglobulin, labeled with biotin, is used as the secondary antibody. This antibody is available from Vector Laboratories, Inc., Burlinga e, CA and is a component of the test kit marketed under the trade designation "Vectastain ABC Kit". These secondary antibodies are preferably diluted with PBS to concentrations ranging from about 5 to about 10 μg / ml and administered
in an amount of 20,000 cells, in amounts ranging from about 0.01 to about 0.5 ml (ie, from about 0.05 μg to about 5.0 μg of the secondary antibody). Enzymatic complexes capable of combining and producing a reservoir to mark the location of antibody binding, which are suitable for use in the present invention, include DH avidin and biotin-labeled horseradish peroxidase H. In a preferred embodiment, an enzymatic complex of avi dina-bio t ina is used. This complex can be prepared by mixing 10 ml of phosphate-buffered saline, 10 mM (PBS: pH 7.5, 0.9% saline, bovine serum albumin 0.1% crystalline grade) with two drops of avidin reagent and two drops of biotin reagent constituting the components of the test kit sold by Vector Laboratories, Inc., under the trade designation "Vectastain ABC Kit" and preferably administered in an amount of 20.00 cells, in amounts ranging from approximately 0.1 until approximately
O .2 (μL). Suitable chromogens and counter-colorants useful in the present invention are the chromogens and counter-ions that produce precipitates, including aminobenzyl diene hydrochloride (DAB) with hematoxylin or 3-amino- 9- et ilcarba zo I (AEC, for its acronym in English) with cosine dye. It is preferable that DAB be used to produce the precipitate and that hematoxylin be used as a counter-colorant, in the practice of the present invention. The measurement of the reactivities of the antibodies within the cell samples, according to the present invention, can be carried out by conventional methods. For example, the measurement or quantification can be carried out through an evaluation by optical microscopy where the intensity of the cellular reactivity is classified on a particular scale (for example, on a scale of 0-4 +) or the intensity The color can be read mechanically using an image analyzer that scans and quantifies the
intensity of the color, providing a comparative result based on a numerical value. Quantification can also be carried out by enzyme-linked immunosorbent assay (ELISA) in which cells fixed to 96-well plates serve as the antigen bound to the solid phase and antibodies (as previously described herein) with a peroxidase conjugate, serve as the assay, in the presence of a chromogen (also described hereinabove). Additionally, the quantification may constitute the quantification of proteins carried out by chemoluminis techniques or procedures or radiolabelling or it may be carried out through an evaluation of a computerized image analysis. The test kit that is used in conjunction with the inventive method herein, preferably contains a quantity of sterile swabs, a vial containing PBS to moisten the swab (s) prior to cell collection, and a second small
containing a conventional tissue culture medium for storing the cells collected during mailing to a central office for analysis. The present invention is described in more detail with reference to the following examples which are presented solely for purposes of illustration and are understood not to indicate or imply any limitation of the extensive invention described herein.
WORK EXAMPLES Used Components
CASE 1: a reagent kit contains: blocking serum (goat); anti-immunoglobulin (goat anti-rabbit) purified by affinity, labeled with biotin (ie, second concentrated antibody); Reagent A (DH avidin); and reagent B (horseradish peroxidase H labeled with biotin), in addition to three mixing bottles (one for the serum or diluted blocking agent, one for the second biotinylated antibody, diluted, and one for the enzyme complex of avidin-bio tina,
diluted), marketed under the trade designation "Vectastain ABC Kit" (product No. PK4005), by Vector Laboratories Inc.
CASE 2: reagent kit with di-aminobenzadine (DAB) substrate for peroxidase, containing: buffer, hydrogen peroxide, DAB, and nickel solution (not used), marketed under the trade name "DAB Substrate kit for Peroxidase" (product No. SK4100), by Vector Laboratories Inc. A solution of DAB is prepared by mixing 10 ml of tap water with 4 drops of buffer, 4 drops of hydrogen peroxide, and 8 drops of DAB.
PRIMARY ANTIBODY N:
a solution of 1 μg / ml of a polyclonal rabbit IgG antibody (capable of detecting the N-terminus regions of the BRCA-1 protein) in DBS. The antibody is marketed under the trade designation "BRCA-1 D-20" (product No. sc641) by Santa-Cruz Biotechnology, Inc.
PRIMARY ANTIBODY C:
a solution of 1 μg / ml of a polyclonal rabbit IgG antibody (capable of detecting the C-region regions of the BRCA-1 protein) in DBS. The antibody is marketed under the trade designation "BRCA-1 C-20" (product No. sc642) by Santa-Cruz Biotechnology, Inc.
SECONDARY ANTIBODY:
a 7.5 μg / ml solution of affinity purified anti-immunoglobulin, labeled with biotin, obtained from CASE 1 (capable of binding rabbit IgG) in PBS.
PBS: 10 mM phosphate-buffered saline, comprising 0.9% saline and 0.1% crystalline grade bovine serum albumin, and having a pH of 7.5.
AVIDINA-BIOTINA;
an enzymatic complex of avi dina-
biotin prepared by mixing 2 drops of avidin reagent (reagent A) and two drops of biotin reagent (reagent B) obtained in CASE 1 with 10 ml of PBS and letting the compiejo form at room temperature for thirty minutes.
Preparation of Cells
A. A number of oral cells were collected from the oral cavities of 15 normal adults, male and female, with cotton swabs moistened with PBS. These adults were in good health and had no history of diseases and their age ranged from 20 to 52 years. The cotton swabs used to collect the cells were placed in separate glass flasks containing an amount of PBS and the flasks were shaken to release the cells from the cotton swabs. Cells released from each probe were counted using a BRIGHTLINEME cytometer (manufactured by Fischer Scientific Co., Pittsburgh, PA) and diluted with PBS to a concentration of 20,000 cells / ml. Then he
deposited the cells with centrifugation, using a CYT0SPIN3MR centrifuge, sold by Shandon, Inc., Lerner Laboratories, 171 Industry Drive, Pittsburgh, PA 15275-1015. The cells deposited by centrifugation were then used to prepare 50 samples, each of which comprised two microscope slides, which contained equal numbers of cells from the same test subject. The cells contained in the pair of prepared slides were then fixed for seven (7) minutes in an acetone / methanol solution 1, 50:50, at -20 ° C and then rinsed twice for 15 minutes in PBS using a mechanical turner for agitation. The rinsed slides were then transferred to a slide tray, which had moistened camera-type clips, which allowed the slides to lie flat so that the reagents could be deposited on the region of each slide, which contained the slides. cells Then the excess PBS was removed from each slide, using a suction pipette, and added,
directly to each slide, over the site where the cells were placed, 200 μl of normal goat blocking agent of 7.5 μg / ml (obtained from CASE 1) in PBS. The slide tray was then covered with plastic wrap and incubated at room temperature for 30 minutes. The excess blocking agent was then removed from each slide using a suction pipette.
B. Samples of normal tissue and tumorous tissue obtained from 35 adult women diagnosed with ovarian cancer were prepared by means of fixation with buffered formalin and paraffin embedding. Once the tissue was embedded in paraffin blocks, the blocks were cut with a microtome, to a thickness of 5 microns. Sections of each test subject were then used to prepare 35 samples, each of which contained two sets of two slides, the first set contained slides that had equal amounts of normal cells and the second set contained slides that had
equal amounts of tumor cells from the same test subject.
Preparation of Samples
An amount of 200 μl of primary antibody N was added, to a slide, in each set of samples, on the regions where the cells were placed. Then an amount of 200 μl of the PRIMARY ANTIBODY C was added to the remaining slide in each set. The slide tray was then covered with plastic wrap and the slides were first incubated for 60 minutes at room temperature and then overnight at 4 ° C. The next day, the temperature of the slides was allowed to reach room temperature for 30 minutes. The slides were then washed twice for 5 minutes with cold PBS. The excess PBS was removed from each slide with a vacuum suction and then an amount of 200 μl of the SECONDARY ANTIBODY was added to each slide, over the
regions where the cells had been placed. The slide tray was then covered with plastic wrapping paper and the slides were incubated for 30 minutes at room temperature. The slides were then removed from the slide tray, placed on glass shelves connected to a mechanical rotor and rinsed for 5 minutes with PBS. The slides were then placed back into the slide tray and the excess PBS was removed with vacuum suction. Then, 4 drops of AVI D INA-B I OT INA were added to each slide, on the regions where the cells had been placed. The slide tray was then covered with plastic wrap and incubated at room temperature for 1 hour. Then the rinsing procedure with PBS was repeated. The excess PBS was then removed from each slide and an amount of 4 drops of DAB solution was immediately added over the regions where the cells had been placed. The slides were then incubated
room temperature for 7 minutes. The DAB solution was then drained from each incubated slide and the slides were rinsed under tap water for 5 minutes. The slides were then stained lightly with counterstain, with hematoxylin, and dehydrated by passing them through solutions that had recent concentrations of alcohol and zilenes. The slides were then drained and coverslips placed over them. The slides were then cured for 1 hour before reading. To preserve the color, the slides were protected from direct light.
Test Methods
Antibody Reactivity (0-4 + scale) - an optical microscope evaluation in which the cells are stained with D-aminobenzadine (DAB) solution prepared in the CASE 2 and stained with hematoxylin counterstain, and where the intensity of color development, which is directly proportional to the intensity of antibody reactivity, is
reads with 200x magnification using a 0-4 + scale for color classification. The colors vary from a faint trace of beige (0 +/-) to an intense chocolate brown (3 + -4 +). The reactivity intensities of the pairs of slides that received the PRIMARY ANTIBODY N and the PRIMARY ANTIBODY C respectively are compared. A difference in reactivity intensities is indicative of a mutation in an allele of BRCA-1 that has resulted in truncation of the protein. Molecular Analysis - a molecular analysis to detect genetic alteration. In this analysis DNA is prepared from the test tissue, analyzed by the polymerase chain reaction (PCR) using sets of primers that amplify the 24 exons of the BRCA-1 gene to obtain products from the PCR The PCR products are then examined for genetic alteration, by analysis by single-strand conformal polymorphism (SSCP, for its acronym in English). These procedures constitute conventional procedures described and detailed in the following
publication: Donna Sha ttuck-E idens et al. , A Col l abora ti ve S? Rvey of 80 Mu t a ti on s. . . , J. Am. Med. Ass'n, Voi. 273 No. 7, February 15, 1995, on pages 535-541.
EXAMPLES FROM 1 TO 85
In the examples from 1 to 85, the cells contained in the slide sets described above were analyzed with respect to the Antibody Reactivity. The results are discussed later. In the examples from 1 to 50, where is. obtained buccal cells from a normal control population, the paired slides in each example, each demonstrating an equal intensity of antibody reactivity. In Examples 51 to 85, where cells were obtained from an experimental population, the slides in eighteen of the examples (where each example comprised two sets of paired slides containing either cells obtained from normal tissue or cells obtained from tumor tissue from the same test subject) demonstrated all
an equal intensity of antibody reactivity. This result suggests the absence of a mutation, either in the cells of normal tissues or in the cells of tumorous tissues. In twelve of the Working Examples, slides containing normal tissue cells showed an equal intensity of antibody reactivity, while slides containing tumorous tissue cells showed a reduced intensity of the reactivity of PRIMARY ANTIBODY C. That The result suggests the absence of a mutation in the cells obtained from normal tissue, and the presence of a non-inherited mutation in the cells obtained from tumorous tissue. In the remaining five Working Examples, the slides containing normal tissue cells demonstrated a 50% reduction in the intensity of the reactivity of the PRIMARY ANTIBODY C while the slides containing tumor tissue cells showed a reduction of 50% or more. 100% in the intensity of the reactivity of the PRIMARY ANTIBODY C. This result suggests the presence of a mutation in the obtained cells, both
of normal tissue as well as tumorous tissue, which is indicative of an inherited mutation. Examples 1 through 85, described above, collectively demonstrate the presence of mutations in BRCA-1, in cell preparations, consistent with an inherited genetic mutation that results in truncation of the protein and, therefore, The susceptibility to breast, ovarian, colon or prostate cancer can be determined in a fast and accurate manner, using the antibody-based assay of the invention.
Examples from 86 to 95
In Examples 86 to 95, 10 sets of paired normal and tumor cell samples, each of which is an example in the group of Working Examples numbered 51 through 85, were selected and subjected to the method of Test by Molecular Analysis described above. The results are tabulated in table 1 below.
SUMMARY OF EXAMPLES 86 to 95
Reactivity of the Molecular Antibody Analysis2
Example No. cells cells cells normal tumorous normal tumorous cells
86 87
89 90 - - + + 91 - + - + 92 - + + + 93 - + + + 94 + + + + 95 '+ + + + "+" = difference in the intensities of the observed reactivity "-" = no observed difference "+" = genetic alteration was identified "-" = no genetic alteration was identified
Examples 94 and 95 show that a positive result in the method or test
Inventive, as described herein, can be considered a definitive proof that the individual analyzed carries a mutated allele. Examples from 86 to 95 collectively demonstrate that the results offered by the inventive method of the present are confirmed by molecular analysis in 80% of the examples performed. discrepancies (eg, false negative readings) shown by examples 90, 92 and 93 can be explained by the fact that not all base mutations result in a change of the encoded amino acid, leaving the protein sequence unchanged. These discrepancies can also be explained by the fact that the mutations may not result in an internal trans-location of the reading frame and / or premature termination, which would also have left the protein sequence unchanged.
It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.
Claims (22)
1. An antibody-based method for determining the presence of the mutated BRCA protein in cells expressing the protein, wherein the non-mutated BRCA protein contains regions of the term amino and the term carboxy and wherein the mutated BRCA protein is typically a protein Reduced or shortened which has lost the regions of the carboxy terminus, the method is characterized in that it comprises: preparing a first and a second equal amount of cells expressing the BRCA protein, wherein the first and the second quantity of cells is obtained from a subject test; administering an amount of a first primary antibody capable of detecting or reacting with the amino terminus regions of the BRCA protein for the first number of cells; administer an equal amount of a second primary antibody capable of detecting or reacting with the carboxy terminus regions of the BRCA protein for the second number of cells; measuring the reactivity of the antibody in the first and second number of cells; and compare the measured reactivities. of the antibody, to determine the presence of the mutated BRCA protein, in the first and second number of cells, wherein a difference in the measured reactivities of the antibody indicates the presence of the mutated protein in the first and second number of cells.
2. The method according to the rei indication 1, characterized in that it comprises: administering equal amounts of a secondary antibody capable of binding to the antibodies, for the first and second quantity of cells containing, either the first or second primary antibody; adding equal amounts of an enzyme complex capable of labeling the binding sites of the antibody, for the first and second number of cells; and later, dyeing and countertening the first and second quantity of cells, before measuring the reactivity of the antibody.
3. The method according to claim 1, characterized in that the cells expressing the BRCA protein are epithelial cells.
4. The method according to claim 3, characterized in that the epithelial cells expressing the BRCA protein are obtained from a non-invasive source of epithelial cells.
5. The method according to claim 4, characterized in that the cells expressing the BRCA protein are buccal cells.
6. The method according to claim 1, characterized in that the BRCA protein is the BRCA-1 protein, and because the first primary antibody is an antibody prepared by immunization, using a sequence peptide representative of amino acids 2-21 of the BRCA-1 protein.
7. The method according to claim 6, characterized in that the first primary antibody is a rabbit polyclonal IgG antibody.
8. The method according to the rei indication 1, characterized in that the BRCA protein is the BRCA-1 protein, and because the second primary antibody is an antibody prepared by immunization using a peptide sequence representative of the amino acids 1843-1862 of the BRCA-protein 1.
9. The method according to claim 8, characterized in that the second primary antibody is a rabbit polyclonal IgG antibody.
10. The method according to claim 1, characterized in that the BRCA protein is the BRCA-2 protein, and because the first primary antibody is an antibody prepared by immunization using a peptide sequence representative of amino acids 3-19 of the BRCA-2 protein.
11. The method according to claim 10, characterized in that the first primary antibody is a goat polyclonal IgG antibody.
12. The method according to the rei indication 1, characterized in that the BRCA protein is the BRCA-2 protein, and because the second primary antibody is an antibody prepared by immunization using a peptide sequence representative of amino acids 3404-3418 of the BRCA-protein 2.
13. The method according to claim 12, characterized in that the second primary antibody is a goat polyclonal IgG antibody.
14. The method according to claim 2, characterized in that the BRCA protein is the BRCA-1 protein, and because the secondary antibody comprises the antiserum obtained from goats immunized with rabbit immunoglobulin.
15. The one in accordance with rei indication 14, characterized in that the secondary antibody comprises an immuno-globulin purified by affinity and labeled with biotin.
16. The method according to claim 2, characterized in that the BRCA protein is the BRCA-2 protein, and because the secondary antibody comprises the antiserum obtained from rabbits immunized with goat immunoglobulin.
17. The method according to claim 2, characterized in that the enzyme complex is selected from the group consisting of avidin DH and horseradish peroxidase H, labeled with biotin.
18. The method according to claim 17, characterized in that the Enzymatic complex comprises an enzymatic complex of avidin-bio tina.
19. The method according to claim 2, characterized in that the first and second quantity of cells are stained with a chromogen producing precipitate, selected from the group consisting of di-aminobenzidine tetrachlorohydrate and 3-amino- 9- et i 1 carbaz ol, and because the first and second numbers of cells are counterstained with a dye selected from the group consisting of the hematoxylin dye and the eos ina dye.
20. The method according to claim 19, characterized in that the aforementioned producing chromogen is di-aminobenz idine tetrachlorohydrate, and because the counter-colorant is the hematoxylin dye.
21. The method according to claim 1, characterized in that the reactivity of the antibody in the first and second amount of cells, is measured using: a method of evaluation by optical microscopy; and an enzyme-linked immunosorbent assay; the quantification of proteins by radiolabeled or chemolumini scenci a; or an evaluation by computerized image analysis.
22. An antibody-based method for determining the presence of mutated BRCA protein in buccal cells, wherein the non-mutated protein contains regions of the amino terminus and the carboxy terminus and because the mutated BRCA protein is typically a reduced or shortened protein that has lost regions of the carboxy terminus, the method is characterized in that it comprises: preparing a first and a second equal amount of cells expressing the BRCA protein, wherein the first and the second equal amount of cells are obtained from a test subject; administering an amount of a first primary antibody capable of detecting or reacting with the amino-terminus regions of the BRCA protein for the first amount of cells; administering an equal amount of a second primary antibody capable of detecting or reacting with the carboxy terminus regions of the BRCA protein for the second number of cells; measuring the reactivity of the antibody in the first and second number of cells; and comparing the measured reactivities of the antibodies, to determine the presence of the mutated protein in the first and second number of cells; wherein a difference in the measured reactivities of the antibodies indicates the presence of the mutated BRCA protein in the first and second number of cells.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
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US042337 | 1987-04-24 |
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MXPA99008774A true MXPA99008774A (en) | 2001-06-26 |
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