U.S. patent application number 11/702273 was filed with the patent office on 2007-06-14 for methods for detecting deficient cellular membrane tightly bound magnesium for disease diagnoses.
This patent application is currently assigned to Magnesium Diagnostics, Inc.. Invention is credited to Ibert C. Wells.
Application Number | 20070134738 11/702273 |
Document ID | / |
Family ID | 23011495 |
Filed Date | 2007-06-14 |
United States Patent
Application |
20070134738 |
Kind Code |
A1 |
Wells; Ibert C. |
June 14, 2007 |
Methods for detecting deficient cellular membrane tightly bound
magnesium for disease diagnoses
Abstract
This invention relates to methods for detecting the deficiency
of magnesium tightly bound to cellular membranes, i.e., magnesium
binding defect, which deficiency is associated with certain
abnormal physiological states, e.g., salt-sensitive essential
hypertension or Type 2 diabetes mellitus.
Inventors: |
Wells; Ibert C.; (Omaha,
NE) |
Correspondence
Address: |
STINSON MORRISON HECKER LLP;ATTN: PATENT GROUP
1201 WALNUT STREET, SUITE 2800
KANSAS CITY
MO
64106-2150
US
|
Assignee: |
Magnesium Diagnostics, Inc.
|
Family ID: |
23011495 |
Appl. No.: |
11/702273 |
Filed: |
February 5, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10053669 |
Jan 24, 2002 |
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11702273 |
Feb 5, 2007 |
|
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09265690 |
Mar 10, 1999 |
6372440 |
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10053669 |
Jan 24, 2002 |
|
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Current U.S.
Class: |
435/7.2 |
Current CPC
Class: |
G01N 2800/321 20130101;
G01N 33/6893 20130101; G01N 2800/042 20130101; Y10S 514/866
20130101 |
Class at
Publication: |
435/007.2 |
International
Class: |
G01N 33/567 20060101
G01N033/567; G01N 33/53 20060101 G01N033/53 |
Claims
1. A method for detecting sodium-sensitive, essential hypertension
in an individual comprising: a. measuring the level of peptide in a
sample of body fluid of said individual, wherein said peptide
consists essentially of the amino acid sequence set forth in SEQ ID
NO:1; and b. comparing said measured level of peptide to a
standard, wherein the standard represents the average level of said
peptide in normal body fluid, whereby, a significantly lower level
of said peptide in the sample is indicative of said
sodium-sensitive, essential hypertension.
2. The method of claim 1 wherein the level of said peptide in said
sample is measured by using an immunological assay.
3. The method of claim 2 wherein said immunological assay utilizes
an antibody to said peptide.
4. The method of claim 2 wherein said immunological assay is an
enzyme-linked immunosorbent assay.
5. A method for monitoring progress in treatment of
sodium-sensitive, essential hypertension in an individual,
comprising: a. measuring the level of peptide in a sample of body
fluid of said individual, wherein said peptide consists essentially
of the amino acid sequence set forth in SEQ ID NO:1; b. treating
the sodium-sensitive, essential hypertension in the individual; c.
repeating step a; and d. comparing said level of peptide of step a,
to the level of said peptide of step c, whereby a significant
increase in the level of said peptide after treatment is indicative
of the progress of treatment of said individual.
6. A method for detecting sodium-sensitive, essential hypertension
in an individual comprising: a. measuring the level of peptide in a
sample of body fluid of said individual, wherein said peptide
consists essentially of the amino acid sequence set forth in SEQ ID
NO:4; and b. comparing said measured level of peptide to a
standard, wherein the standard represents the average level of said
peptide in normal body fluid, whereby, a significantly lower level
of said peptide in the sample is indicative of said
sodium-sensitive, essential hypertension.
7. The method of claim 6 wherein the level of said peptide in said
sample is measured by using an immunological assay.
8. The method of claim 7 wherein said immunological assay utilizes
an antibody to said peptide.
9. The method of claim 7 wherein said immunological assay is an
enzyme-linked immunosorbent assay.
10. A method for monitoring progress in treatment of
sodium-sensitive, essential hypertension in an individual,
comprising: a. measuring the level of peptide in a sample of body
fluid of said individual, wherein said peptide consists essentially
of the amino acid sequence set forth in SEQ ID NO:4; b. treating
the sodium-sensitive, essential hypertension in the individual; c.
repeating step a; and d. comparing said level of peptide of step a,
to the level of said peptide of step c, whereby a significant
increase in the level of said peptide after treatment is indicative
of the progress of treatment of said individual.
Description
CROSS REFERENCE
[0001] This application is a continuation of U.S. patent
application having Ser. No. 10/053,669, filed Jan. 24, 2002, which
is a continuation of U.S. patent application having Ser. No.
09/265,690, filed Mar. 10, 1999, the disclosures of which are
incorporated in full herein.
BACKGROUND OF THE INVENTION
[0002] 1. Field of Invention
[0003] This invention relates to methods and diagnostic kits for
detecting the cellular membrane magnesium binding defect, which
deficiency is critically associated with certain abnormal
physiological states, e.g. sodium-sensitive essential hypertension
and type 2 insulin-resistant diabetes mellitus.
[0004] 2. Description of the Prior Art
[0005] Elevated arterial pressure, namely hypertension, is probably
among the most important public health problems in developed
countries. It is of common occurrence, asymptomatic, readily
detectable, and often leads to lethal complications if not treated.
Although there are exceptions, most untreated adults with
hypertension will continue to experience further increases in their
arterial pressure over time. Reports based on actuarial data and
clinical experience, estimate that untreated hypertension shortens
life by 10 to 20 years. This lower life expectancy is believed to
be due to an acceleration of the atherosclerotic process, with the
rate of acceleration related in part to the severity of the
hypertension. Even individuals with relatively mild disease--those
individuals without evidence of end-organ damage--if left untreated
for 7 to 10 years have a high risk of developing significant
complications, and more than 50 percent of them will ultimately
experience end-organ damage related to hypertension. End organ
damage can include cardiomegaly, congestive heart failure,
retinopathy, a cerebrovascular accident, and/or renal
insufficiency. Thus, even in its mild forms, hypertension can be a
lethal disease, if left untreated.
[0006] Although awareness of the problems associated with elevated
arterial pressure has increased, in 90 to 95 percent of the 60
million, minimally estimated, existing cases in the United States,
the cause of the disease, and thus potentially its prevention and
cure, is still largely unknown. These individuals have only
generalized or functional abnormalities associated with their
hypertension and are often diagnosed as having primary, idiopathic
or essential hypertension. Several abnormalities have been
identified in patients with essential hypertension (see e.g., Meyer
P and Marche P, Am. J. Med. Sci. 295: 396-399 (1988)), often with
claims, later contested or unsubstantiated, of the abnormalities
being primarily responsible for the hypertension. This situation
has been attributed generally to the likely possibility that
essential hypertension has more than one cause, each of which may
be a set of genetically determined, contributory abnormalities,
which in turn interact with environmental factors.
[0007] The most widely recognized of these possible causes of
essential hypertension is sodium, i.e. sodium ion (Na+)sensitivity,
also commonly referred to as salt (NaCl)-sensitivity. In such
patients hypertension is exacerbated by a high dietary salt intake
and diminished by dietary salt restriction. It has been assumed
that this abnormality reflects cellular membrane defect, and that
this defect occurs in many, perhaps all, cells of the body,
particularly the vascular smooth muscle cells. Based on studies
using erythrocytes, this defect has been estimated to be present in
35 to 50 percent of the essential hypertension population.
[0008] However, Applicant discovered, as disclosed below, the
actual complex mechanism underlying sodium-sensitive hypertension,
which discovery has yielded a new diagnostic methodology to detect
this disease at its early stages.
[0009] Type 2 diabetes mellitus is the most common form of diabetes
mellitus, comprising 85-90% of the diabetic population and taking
heterogeneous forms. Overt diabetes characteristically appears
after the age of 40, has a high rate of genetic penetrance
unrelated to HLA genes, and is associated with obesity. A strong
hereditary component is evident. For example, concordance rates in
identical twins is nearly 100 percent.
[0010] Among American whites the estimated incidence of Type 2
diabetes mellitus in 1976 was between 1 and 2 percent, but the
prevalence has risen as the population has aged and become more
obese. More than 10 percent of the older population now suffers
from the disease. According to the 1990-1992 National Health
Interview Survey, about 625,000 cases of diabetes are diagnosed in
the United States each year--more than 6 times the 1935-36
rate.
[0011] Many consider insulin resistance to be the primary cause of
Type 2 diabetes mellitus. This pathological state and the
consequent hyper-insulinemia develop years before insulin secretion
diminishes and overt diabetes mellitus is present. About 20 percent
of the white population of the United States has impaired glucose
tolerance, i.e.hyperglycemia--the virtually universally accepted
sign of the presence of diabetes mellitus.
[0012] Patients affected with overt Type 2 diabetes mellitus retain
some endogenous insulin-secreting capacity, but insulin levels in
plasma are low relative to the magnitude of insulin resistance and
ambient plasma glucose levels. Such patients do not depend on
insulin for immediate survival and rarely develop diabetic
ketosis.
[0013] The clinical presentation of Type 2 diabetes mellitus is
insidious. The classical symptoms of diabetes may be mild and
tolerated for a long time before the patient seeks medical
attention. Moreover, if hyperglycemia is asymptomatic, the disease
becomes clinically evident only after complications develop. Such
complications include atherosclerosis, the risk for which is
greatest in poorly controlled patients. Other sequela of diabetes
mellitus are myocardial infarction, stroke, peripheral vascular
disease and lower extremity gangrene, neuropathy, nephropathy,
diabetic foot syndrome, cardiomyopathy and dermopathy.
[0014] Little is known about the specific genetic abnormalities
associated with most forms of Type 2 diabetes mellitus. However,
applicant has observed the highly frequent occurrence of the
magnesium binding defect in the erythrocyte membranes of mildly
affected Type 2 diabetics.
[0015] Others have found insulin resistance to be caused by what
was ostensibly the magnesium binding defect. (See Mattingly M T,
Brzezinske W A, Wells I C, Clin. Exper. Hypertension--Theory and
Practice A13: 65-82 (1991).
[0016] These observations strongly support the concept that the
magnesium binding defect, which is genetic, is the cause of insulin
resistance. Therefore, the detection of the presence of this defect
in an individual who is asymptomatic would indicate the presence of
Type 2 diabetes mellitus in its earliest stage so that management
of the disease could begin at the earliest possible time.
SUMMARY OF THE INVENTION
[0017] This invention involves methods for the detection in humans
of physiological disorders, such as sodium-sensitive, essential
hypertension and adult onset, Type 2, insulin resistant diabetes
mellitus, for which the subnormal binding of magnesium to cellular
membranes of the somatic cells is a contributory, critical cause.
These methods comprise the quantification of the concentrations, in
blood plasma from the above individuals, of the polypeptide
degradation products derived from the amidated C-terminal region of
the tachykinins such as Substance P. These degradation products are
embodied in the amino acid sequence of the pentapeptide which
characterizes the amidated, C-terminal amino acid sequences of all
of the tachykinins, of mammalian origin, i.e. Phe-X(Phe,
Val)-Gly-Leu-Met-NH.sub.2.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0018] Applicant quantified the magnesium contents of the several
anatomical compartments of whole blood from some essential
hypertension patients and normotensive control subjects and
discovered several differences. The most noteworthy difference was
the decreased levels of magnesium firmly bound to the erythrocyte
membranes of the hypertensive patients. The magnitude of this
previously unknown defect correlated positively with the magnitude
of the decreased concentration of intracellular magnesium which in
turn correlated positively with the average blood pressures of the
patients. Further, another novel observation was that this
magnesium binding defect was corrected by incubating the
erythrocytes from the hypertensive patients with blood plasma from
the control subjects (Mattingly M T, Brezezinski W A, Wells I C,
Clin. Exper. Hypertension-Theory and Practice A13: 65-82
(1991)).
[0019] This investigation was extended to include two strains of
genetic, sodium-sensitive, hypertensive rats, i.e. the SHR and
SS/Jr rats, and their respective normotensive controls, i.e. the
WKY and SR/Jr rats (Wells I C and Agrawal D K, Can. J. Physiol.
Pharmacol. 70: 1225-1229 (1992)). The magnesium binding defect was
also observed to occur in these two strains of hypertensive rats as
well as in the SR/Jr normotensive strain. It was concluded from
these observations that the magnesium binding defect could only be
a contributory, though perhaps a critical, cause of hypertension
generation. Other investigators have collected evidence to indicate
that in these two hypertensive rat strains the enzyme systems
required for the extrusion of excess sodium ion from the cells,
i.e. the Na.sup.+, K.sup.+-ATPase and/or the Na.sup.+,
K.sup.+-cotransport enzyme, are defective and that the passive
permeability of the cell membranes for sodium ion of the SHR rat is
greater than that of the control WKY rat.
[0020] Accordingly, Applicant made several conclusions about the
mechanisms of hypertension generation in the SHR and SS/Jr rats
(both of which exhibit sodium-sensitive hypertension). First, the
magnesium binding defect in the cellular membrane of the vascular
smooth muscle cell, for example, and perhaps those of all somatic
cells, permits per unit of time more than the normal amounts of
sodium ion to enter passively into the cell even though the
extracellular concentration of this ion is normal. Second, because
the enzyme systems which remove excess sodium ion from the cell are
defective, the intracellular sodium ion concentration increases to
above normal levels. Third, because the extracellular sodium ion
concentration tends to remain greater than the intracellular
concentration, the sodium-calcium exchange enzyme within the cell
membrane begins to export sodium ion from the cell and to import
calcium ion. Fourth, the resulting increased intracellular calcium
ion concentration stimulates the smooth muscle to contract. When
the vascular smooth muscle contracts, the lumens of the arterioles
in the peripheral circulation decrease in diameter thereby
increasing the resistance to blood flow. Finally to overcome this
increased resistance to blood flow, the heart must contract more
strongly and this increased force is reflected as increased blood
pressure.
[0021] As indicated above, the normotensive SR/Jr rat also has the
magnesium binding defect. However, this rat is normotensive and can
tolerate greatly elevated levels of dietary NaCl ostensibly because
its sodium ion extrusion enzymes adequately prevent an increase in
the intracellular concentration of this ion.
[0022] The results of further experimentation employing the two
sodium-sensitive hypertensive rat strains are as follows. The total
intracellular concentrations of sodium, potassium and calcium in
the sodium-sensitive, hypertensive SHR and SS/Jr rats, as compared
to those of the normotensive WKY and SR/Jr rats, are entirely
consistent with the above postulated mechanism, i.e. elevated
concentrations of sodium and calcium, decreased concentration of
potassium. The substances in normal human and rat plasmas that
correct the magnesium binding defect in erythrocyte membranes were
identified as the pentapeptide Phe-Phe-Gly-Leu-Met-NH.sub.2 (SEQ ID
NO:1) and the tetrapeptide Phe-Gly-Leu-Met-NH.sub.2 (SEQ ID NO:2)
which occur at the C-terminal end of the tachykinin Substance P
whose amino acid sequence is
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH.sub.2 (SEQ ID
NO:3).
[0023] Evidence was obtained to indicate that the generalized
C-terminal sequence of the tachykinins Phe-X(Phe,
Val)-Gly-Leu-Met-NH.sub.2 (SEQ ID NO:4) embodies the substances in
normal plasma which prevent the magnesium binding defect in
cellular membranes. This defect plays a critical role in the
genesis of sodium-sensitive hypertension and Type 2 diabetes
mellitus. Further, the intravenous administration of the
pentapeptide of SEQ ID NO:1 to the sodium sensitive SS/Jr rat not
only corrected the magnesium binding defect in its erythrocytes but
also reduced its systolic blood pressure from an elevated value
of210 mm Hg to a normal value than 160 mm Hg.
[0024] It is apparent that an essential hypertensive person in whom
the magnesium binding defect exists is, to a very high degree of
probability, a sodium-sensitive hypertensive and that the
restriction of the dietary intake of sodium chloride (and other
sources of sodium ion) by this individual would be therapeutically
beneficial. Contrariwise, an essential hypertensive person without
the magnesium binding defect is in all probability a
sodium-insensitive hypertensive. Not only would such a person
suffer needlessly if restricted to the minimum dietary sodium
chloride intake consistent with a healthy existence but there is
evidence to indicate that such a diet would be harmful for certain
ones of these essential hypertensive persons.
[0025] It is further apparent that a method that would permit the
rapid and accurate determination of the blood plasma levels of the
polypeptides which have the amino acid sequences corresponding to
those of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:4 all of which are
equally active in preventing or correcting the magnesium binding
defect, would be of value to a clinician for the diagnosis and
treatment of essential hypertension. It is realistic to expect that
if the blood plasma levels of these compounds are subnormal in an
essential hypertensive patient, the magnesium binding defect is
present and that the hypertension can be classified as
sodium-sensitive and can therefore be treated appropriately. On the
other hand, if the concentrations of these substances are at least
normal, then the magnesium binding defect is not present and the
hypertension belongs to the salt-insensitive classification. Also,
applicant has observed the highly frequent occurrence of the
magnesium binding defect in the erythrocyte membranes of mildly
affected Type 2 diabetics, as discussed above, and analogous
considerations would apply for the diagnosis of this disease.
[0026] A most preferred embodiment of this invention employs
immunochemical procedures (i.e., "binding assays") to detect the
occurrence of the magnesium binding defect. This invention involves
binding assays wherein a binding pair member having affinity to one
or more of the polypeptides listed above is employed to detect the
amount(s), presence or absence of the polypeptide(s) in question in
blood plasma. A preferred binding pair member is an antibody. Both
direct and indirect procedures are used with known or constant, but
unknown, concentrations of polypeptide (analyte) and antibody to
determine the quantitative relationship between the two substances.
This calibration procedure requires that either the analyte or
antibody be labeled, either before or subsequent to binding, with
an easily and accurately quantifiable material so that from the
quantity of label present after the binding procedure and the
previously determined binding relationship, the amount of analyte
initially subjected to the binding reaction can be determined with
acceptable accuracy. This methodology is widely known and
extensively utilized because the extreme sensitivity possible with
this method allows the quantification of physiological important,
low molecular weight analytes such as steroid hormones, e.g.
progesterone, in biological fluids, e.g. blood plasma, at
concentrations in the picogram, i.e. 10.sup.-12 grams, per mL
range. The construction and utilization of various such assay
systems can be accomplished by one skilled in the art. Examples of
such systems are described and discussed in detail in An
Introduction to Radioimmunoassay and Related Techniques, fourth
edition, by T. Chard: 1990; Elsiever Science Publishing Co., Inc.;
New York which reference is incorporated herein.
[0027] For the purpose of illustration, the construction of a
suitable assay system for the determination of the concentrations
of the polypeptides of interest in blood plasma is as follows.
Since each of the polypeptides above has only one, and the same,
immunological combining site, namely the pentapeptide of SEQ ID
NO:1, suitable modifications of it will be used for labeling with a
label such as the radioisotope, iodine-125, and for the raising of
the necessary antibody. SEQ ID NO:1, its analog in which one of the
phenylalanine (Phe) residues is replaced with a tyrosine (Tyr)
residue, and its deamidated product are available from commercial
sources, e.g. (Sigma Chemical Co., St. Louis, Mo.). The Tyr analog
is labeled with iodine-125 by procedures described in the above
reference and is used as the "trace analyte". The deamidated
peptide is conjugated with a carrier protein, as described below
for use in the production in an animal of a polyclonal antibody
having a high titer against the peptide.
[0028] Briefly, a polyclonal antibody is prepared by immunizing an
animal with an immunogenic protein or polypeptide and collecting
antisera from that immunized animal. A wide range of animal species
is used for the production of antisera, and the choice is based on
the phylogenetic relationship to the antigen. Typically the animal
used for production of antisera is a rabbit, a guinea pig, a
chicken, a goat, or a sheep. Because of the relatively large blood
volumes of sheep and goats, these animals are preferred choices for
production of large amounts of polyclonal antibodies.
[0029] As is well known in the art, antigenic substances may vary
in their abilities to generate an immune response. It is necessary
in this case, therefore, to boost the host immune system by
coupling such weak immunogens (e.g., a peptide or polypeptide) to a
carrier, which is recommended in the present case. Examples of
common carriers are keyhole limpet hemocyanin ("KLH", which is
preferred in this case) and bovine serum albumin (BSA). Means for
conjugating a polypeptide to a carrier protein are well known in
the art and include the use of MBS
(m-malecimidobenzoyl-N-hydroxysuccimide ester), EDAC
(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), and
bisdiazotized benzidine.
[0030] The conjugation and antibody production services are also
available commercially (e.g., from Rockland, Immunochemicals for
Research, Gilbertsville, Pa.). The pentapeptide SEQ ID NO:1 is used
as the analyte standard.
[0031] As is also well known in the art, the immunogenicity of a
particular immunogen can be enhanced by the use of non-specific
stimulators of the immune response, known as adjuvants. Cytokines,
toxins or synthetic compositions may also be used as adjuvants. The
most commonly used adjuvants include complete Freund's adjuvant (a
non-specific stimulator of the immune response containing killed
Mycobacterium tuberculosis) and incomplete Freund's adjuvant which
does not contain the bacteria.
[0032] Milligram quantities of antigen (immunogen) are preferred
although the amount of antigen administered to produce polyclonal
antibodies varies with the nature and composition of the immunogen
as well as with the animal used for immunization. A variety of
routes can be used to administer the immunogen (subcutaneous,
intramuscular, intradermal, intravenous and intraperitoneal). The
production of polyclonal antibodies may be monitored by sampling
blood of the immunized animal at various times following
inoculation.
[0033] A second, booster injection, may also be given. The process
of boosting and titering is repeated until a suitable titer is
achieved. When a desired level of immunogenicity is obtained, the
immunized animal can be bled and the serum isolated and stored,
and/or the animal can be used to generate monoclonal antibodies
(MAbs).
[0034] For production of rabbit polyclonal antibodies, the animal
can be bled through an ear vein or alternatively by cardiac
puncture. The removed blood is allowed to clot and then centrifuged
to separate serum components from whole cells and blood clots.
Sterility is maintained throughout this preparation. The serum may
be used as such for various applications or else the desired
antibody fraction may be isolated and purified by well-known
methods, such as affinity chromatography using another antibody, a
peptide bound to a solid matrix, or by using, e.g., protein A or
protein G chromatography.
[0035] Instead of using polyclonal antibodies, monoclonal
antibodies (MAbs) can be used in the practice of this invention.
MAbs may be readily prepared through use of well-known techniques,
such as those exemplified in U.S. Pat. No. 4,196,265, incorporated
herein by reference. Typically, this technique involves immunizing
a suitable animal with a selected immunogen, e.g., a purified or
partially purified protein, polypeptide, peptide or domain. The
immunizing substance is administered in a manner effective to
stimulate antibody producing cells.
[0036] The methods for generating monoclonal antibodies (MAbs)
generally begin along the same lines as those for preparing
polyclonal antibodies. Rodents such as mice and rats are preferred
animals; however, the use of rabbit, sheep, or frog cells is also
possible. The use of rats may provide certain advantages (Goding,
In: Monoclonal Antibodies: Principles and Practice, 2d ed., 1986,
pp. 60-61), but mice are preferred, with the BALB/c mouse being
most preferred as it is routinely used and generally gives a higher
percentage of stable fusions.
[0037] The animals are injected with antigen, generally as
described above. The antigen may be coupled to carrier molecules
such as keyhole limpet hemocyanin if necessary. The antigen is
typically mixed with adjuvant, such as Freund's complete or
incomplete adjuvant. Booster injections with the same antigen are
made at approximately two-week intervals.
[0038] Following immunization, somatic cells with the potential for
producing antibodies, specifically B lymphocytes (B cells), are
selected for use in the MAb generating protocol. Antibody-producing
B cells are usually obtained by disbursement of the spleen, but
tonsil, lymph nodes, or peripheral blood may also be used. Spleen
cells are preferred because they are a rich source of
antibody-producing cells that are in the dividing, plasmablast
stage.
[0039] The antibody-producing B lymphocytes from the immunized
animal are then fused with cells of an immortal myeloma cell line,
generally one from the same species as the animal that was
immunized. Myeloma cell lines suited for use in hybridoma-producing
fusion procedures preferably are non-antibody-producing, have high
fusion efficiency, and enzyme deficiencies that render them
incapable of growing in certain selective media which support the
growth of only the desired fused cells (hybridomas). Any one of a
number of myeloma cells may be used, as is known to those of skill
in the art (Goding, pp. 65-66, 1986).
[0040] Methods for generating hybrids of antibody-producing spleen
or lymph node cells and myeloma cells usually comprise mixing
somatic cells with myeloma cells in about a 2:1 proportion in the
presence of an agent or agents (chemical or electrical) that
promote the fusion of cell membranes. The original fusion method
using Sendai virus has largely been replaced by those using
polyethylene glycol (PEG), such as 37% (v/v) PEG, as has been
described in the art. The use of electrically induced fusion
methods is also appropriate.
[0041] Fusion procedures usually produce viable hybrids at low
frequencies. However, this does not pose a problem, as the viable,
fused hybrids are differentiated from the parental, unfused cells
(particularly the unfused myeloma cells that would normally
continue to divide indefinitely) by culturing in a selective growth
medium. The selective medium is generally one that contains an
agent that blocks the de novo synthesis of nucleotides. Exemplary
and preferred agents are aminopterin, methotrexate, and azaserine.
Aminopterin and methotrexate block de novo synthesis of both purine
and pyrimidine nucleotides, whereas azaserine blocks only de novo
nucleotide purine synthesis. Where aminopterin or methotrexate is
used, the media is supplemented with hypoxanthine and thymidine as
a source of nucleotides (HAT medium) by salvage pathways. Where
azaserine is used, the media is supplemented with hypoxanthine.
[0042] A preferred selection medium is HAT. Only cells capable of
operating nucleotide salvage pathways are able to survive in HAT
medium. The myeloma cells are defective in key enzymes of the
salvage pathway, e.g., hypoxanthine phosphoribosyl transferase
(HPRT), and therefore, they cannot survive. The B cells can operate
this pathway, but they have a limited life span in culture and
generally die within about two weeks. Therefore, the only cells
that can survive in the selective media are those hybrids formed
from myeloma and B cells.
[0043] This culturing provides a population of hybridomas from
which particular clones are selected. The selection of hybridomas
is performed by culturing the cells in microtiter plates, followed
by testing the individual clonal supernatants (after about two to
three weeks) for antibody producers using ELISA IgG assays.
Antibody positive hybridomas are screened further for MAbs with
desired reactivity using antigen based assays. Such assays are
normally sensitive, simple, and rapid, such as radioimmunoassays,
enzyme immunoassays, dot immunobinding assays, and the like.
[0044] The selected hybridomas are then serially diluted and cloned
into individual antibody-producing cell lines, clones of which are
then propagated indefinitely to provide MAbs. The cell lines can be
exploited for MAb production in two basic ways.
[0045] A sample of the hybridoma can be injected (often into the
peritoneal cavity) into a histo-compatible animal of the type that
was used to provide the somatic and myeloma cells for the original
fusion (e.g., a syngenetic mouse). Optionally, the animals are
primed with a hydrocarbon, especially oils such as pristane
(tetramethylpentadecane) prior to injection. The injected animal
develops tumors secreting the specific monoclonal antibody produced
by the antibody producing hybridoma. The ascites fluid of the
animal, and in some cases blood, can then be obtained to provide
MAbs in high concentration.
[0046] The individual cell lines could also be cultured in vitro;
where the MAbs are naturally secreted into the culture medium from
which they can be readily obtained in high concentrations.
[0047] MAbs produced by either means may be further purified, if
desired, using filtration, centrifugation and various
chromatographic methods such as HPLC or affinity
chromatography.
[0048] Monoclonal antibodies are preferred since the hybridoma
cells which produce them can be kept in vitro indefinitely, and the
assay can be more accurate due to the higher selectivity that can
be achieved with a monoclonal antibody assay. The raising of
monoclonal antibodies is well known. Means for preparing and
characterizing antibodies are also well known in the art. (See,
e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, 1988, which is incorporated herein by reference.)
[0049] Whether a monoclonal or polyclonal antibody is employed in
the practice of this invention, the steps involved in carrying out
an assay consistent with the teachings of this invention are the
same. Broadly speaking, the assay of this invention involves first
constructing a standard curve involving known concentrations and
amounts of reagents that subsequently can be used in assays where
the concentration of the polypeptide associated with the magnesium
binding defect is being determined in plasma or other body fluid
samples. These techniques, while not previously practiced in
connection with this specific polypeptide, are otherwise known in
the art as having been practiced in the detection of other
analytes.
[0050] However, in the context of this invention, an assay can be
practiced as follows. One way to construct an assay is to use the
radioimmunoassay ("RIA"). As is known in the art, the RIA is an
analytic technique which depends on the competition (affinity) of
an antigen for antigen-binding sites on antibody molecules.
Standard curves are constructed from data gathered from a series of
samples each containing the same known concentration of labeled
antigen, and various, but known, concentrations of unlabeled
antigen. Antigens are labeled with a radioactive isotope tracer.
The mixture is incubated in contact with an antibody. Then the free
antigen is separated from the antibody and the antigen bound
thereto i.e., the antibody-antigen complex. Finally, by use of a
suitable detector, such as a gamma or beta radiation detector, the
percent of either the bound or free labeled antigen or both is
determined. This procedure is repeated for a number of samples
containing various known concentrations of unlabeled antigens and
the results are plotted as a standard graph. The percentages of
bound tracer antigens are plotted as a function of the antigen
concentration. Typically, as the total antigen concentration
increases the relative amount of the tracer antigen bound to the
antibody decreases. After the standard graph is prepared, it is
thereafter used to determine the concentration of antigen in
samples undergoing analysis.
[0051] In an analysis, the sample in which the concentration of
antigen is to be determined is mixed with a known amount of tracer
antigen. Tracer antigen is the same antigen known to be in the
sample but which has been labeled with a suitable radioactive
isotope. The sample with tracer is then incubated in contact with
the antibody. Then it can be counted in a suitable detector which
counts the free antigen remaining in the sample. The antigen bound
to the antibody or immunoadsorbent may also be similarly counted.
Then, from the standard curve, the concentration of antigen in the
original sample is determined.
[0052] The first step in a standard curve is to incubate a fixed
amount of the tracer analyte with a reagent blank and with a series
of dilutions of the antibody in constant volumes of buffer
containing bovine serum albumin ("BSA"). At the end of the
incubations each of the antibody-tracer analyte complexes formed
are precipitated by the addition of constant amounts of
polyethyleneglycol 4000. The level of radioactivity of each
precipitate is determined with the use of a suitable gamma counter.
These values, in descending order, are plotted on the ordinate
(normal scale) of semilog paper against the dilutions of antibody,
in descending order (highest to lowest), plotted on the abscissa
(log scale).
[0053] From this plot is read the dilution of antibody which
combines with 50 percent of the labeled analyte. This particular
combination of concentrations of tracer analyte and antibody is
used for the construction of the standard curve.
[0054] To construct the standard curve, a second incubation is
carried out under the same conditions as before except that the
above dilution of antibody and concentration of tracer analyte are
introduced together into a series of incubation tubes containing
increasing concentrations of the standard analyte. The processes of
incubation, precipitation of the antibody-analyte complexes, and
quantification of the radioactivities are carried out as before.
This time, a plot on semilog paper of the levels of radioactivity
in descending order on the ordinate against the concentrations of
standard analyte in ascending order on the abscissa is prepared and
constitutes in the standard curve.
[0055] To determine the quantity of the polypeptide in a test
sample, a third incubation is carried out using the conditions used
in constructing the standard curve except that the ascending
concentrations of standard analyte are replaced by two or more
suitable dilutions of a concentrate of plasma polypeptides prepared
as indicated above. The concentrations of analyte added to the
incubation tubes are read from the standard curve by noting the
concentration of standard that corresponds to each level of
radioactivity measured for the tubes containing the unknowns. Such
procedures can, of course, be automated, as is known in the
art.
[0056] From the standpoint of good practice the standard curve
should be developed each time unknown samples are assayed.
[0057] Once the conditions for the standard curve have been
fine-tuned or adjusted so that the curve includes the
concentrations of analyte likely to be encountered in a specific
biological fluid, e.g. blood plasma, the standards and reagents,
together with appropriate instructions for use, can be packaged in
a "kit" for commercial distribution.
[0058] The principles involved in the radioimmunoassay system above
can also be applied to a variety of immunoassay systems, preferably
competitive binding assays, of varying degrees of sensitivity for
the quantification of the polypeptides involved in the detection of
the magnesium binding defect. In each case, the antibody can be
monoclonal or polyclonal. It is bound to a support so that the
antibody-analyte complexes can be readily separated from the
incubation mixtures. The labels used for forming the tracer analyte
determine the sensitivities of the systems and provide for
calorimetric (least sensitive), radioactive, fluorometric and
chemiluminescent (most sensitive) endpoints. Consequently, the
conventional apparatuses used for the determination of each type of
endpoint will be required.
[0059] There are many ways the antibody may be bound and the tracer
analyte labeled. For example, the antibody can be bound in the
following ways:
[0060] a) Antibody adsorbed on a polystyrene tube or surface
(microtiter plate). Complexes are isolated by washing.
[0061] b) Antibody adsorbed on a polyvinyl tube or surface
(microtiter plate). Complexes are isolated by washing.
[0062] c) Antibody adsorbed on 6 mm polystyrene spheres. Complexes
are isolated by centrifugation and washing.
[0063] d) Antibody adsorbed on 6 mm polyvinyl spheres. Complexes
are isolated by centrifugation and washing.
[0064] e) Antibody bound to 5 um microparticles of paramagnetic
ferrous oxide. The surfaces of particles are derivitized with a
substance that has a terminal amino group. The antibody is linked
to the surface by the use of glutaraldehyde. Complexes are isolated
by applying a magnetic field to hold the particles against the
surface of the incubation tube, and then washing.
[0065] f) Antibody bound to 5 urn microparticles of paramagnetic
chromium dioxide. Binding of antibody to surface of particles and
isolation of complexes accomplished as described in (e) above.
[0066] g) Antibody bound to sepharose (an insoluble complex
carbohydrate) after the surface of the sepharose is modified by use
of cyanogen bromide. Complexes are isolated by centrifugating and
washing.
[0067] h) Antibody bound to agarose (an insoluble complex
carbohydrate). Binding of antibody and isolation of complexes are
accomplished as in (g) above.
[0068] i) Antibody derivitized with biotin by use of biocytin and
glutaraldehyde. (Biotinyl-N-hydroxysuccimide ester, or
biotinyl-p-nitrophenyl ester, or
caproylamidobiotinyl-N-hydroxy-succimimide ester may also be used
for biotinylation). Complexes are isolated by allowing complex to
combine with the protein avidin which is bound to a solid support
such as plastic spheres, paramagnetic particles, or insoluble
carbohydrates by the methods indicated above.
[0069] In general, the detection of immunocomplex formation is well
known in the art and may be achieved through the application of a
number of tags besides the radioactive tag used in the RIA
described above. These other methods are based upon the detection
of fluorescent, biological, or enzymatic tags, for example. Some
examples include the following:
[0070] 1) Analyte conjugated with horseradish peroxidase using
glutaraldehyde. Quantification is accomplished by: a) measuring
intensity of color produced in the presence of hydrogen peroxide
and o-phenylenediamine or preferably
3,3',5,5'-tetramethylbenzidine; b) measuring fluorescence intensity
after the addition of hydrogen peroxide and fluorescein or
rhodamine; c) by measuring chemiluminescence intensity after the
addition of hydrogen peroxide and luminol plus benzothiazole. The
equipment required includes colorimeter or spectrophotometer
(unaided normal vision sufficient for qualitative assessment),
spectrofluorimeter, or luminometer, respectively.
[0071] 2) Analyte labeled with acridinium ester by use of
4-(2-succinimidyloxy-carbonyl
ethyl)-phenyl-10-methylacridinium-9-carboxylate fluorosulfonate.
Quantification is accomplished by measuring intensity of
chemiluminescence produced by the addition of alkaline hydrogen
peroxide. A luminometer is required.
[0072] 3) Analyte labeled with fluorescein isothiocyanate.
Quantification is accomplished by measuring the intensity of
fluorescence after addition of hydrogen peroxide and a peroxidase
such as horseradish peroxidase. A spectrofluorimeter is
required.
[0073] 4) Analyte labeled with alkaline phosphatase by use of
glutaraldehyde. Quantification is accomplished by measuring
intensity of fluorescence after addition of
4-methylumbelliferylphosphate. A spectrofluorimeter is obviously
required.
[0074] 5) Analyte labeled with rhodamine isothiocyanate.
Quantification is accomplished the same as in (3) above.
[0075] 6) Analyte labeled with glucose-6-phosphate dehydrogenase by
use of glutaraldehyde. Quantification is accomplished by measuring
ultraviolet light absorbed after the addition of
glucose-6-phosphate and nicotinamide-adenine dinucleotide. A UV
spectrophotometer is required.
[0076] 7) Analyte labeled by conjugation with bacterial peroxidase.
Conjugation and quantification is accomplished the same as in (1)
above.
[0077] By use of one of the various bound forms of the antibody and
of labeled forms of analyte described above it is possible to
construct many kinds of competitive binding assay systems for the
quantification of the pentapeptide (SEQ ID NO:1) and its
tetrapeptide degradation product (SEQ ID NO:2) which occur in human
blood plasma and prevent the occurrence of the magnesium binding
defect in cell membranes. Alternatively, in each such system, the
tracer analyte, rather than the antibody, can be the bound member
of the system. Since these systems vary in their sensitivities and
equipment requirements, it is possible to select a system according
to the specific requirements of or adapted to the existing
equipment employed by the user.
[0078] The specific reagents and other requirements for each
system, except hardware, and directions for use can be packaged in
"kits" containing various combinations of the above reagents,
together with the necessary containers containing washing
solutions, for commercial distribution.
[0079] While several embodiments of this invention have been
described, others will readily be apparent to those skilled in the
art. Such embodiments are included in this invention, unless the
claims that follow expressly state otherwise.
Sequence CWU 1
1
4 1 5 PRT Homo sapiens MOD_RES (5)..(5) AMIDATION 1 Phe Phe Gly Leu
Met 1 5 2 4 PRT Homo sapiens MOD_RES (4)..(4) AMIDATION 2 Phe Gly
Leu Met 1 3 11 PRT Homo sapiens MOD_RES (11)..(11) AMIDATION 3 Arg
Pro Lys Pro Gln Gln Phe Phe Gly Leu Met 1 5 10 4 5 PRT Homo sapiens
MOD_RES (5)..(5) AMIDATION VARIANT (2)..(2) "X" may be either Phe
or Val. 4 Phe Xaa Gly Leu Met 1 5
* * * * *