U.S. patent application number 12/713675 was filed with the patent office on 2010-06-17 for pregnancy and sex identification test based on saliva or other bodily fluids.
This patent application is currently assigned to DREAM MAKERS, LLC. Invention is credited to Vito J. D'Aurora.
Application Number | 20100151593 12/713675 |
Document ID | / |
Family ID | 46150274 |
Filed Date | 2010-06-17 |
United States Patent
Application |
20100151593 |
Kind Code |
A1 |
D'Aurora; Vito J. |
June 17, 2010 |
PREGNANCY AND SEX IDENTIFICATION TEST BASED ON SALIVA OR OTHER
BODILY FLUIDS
Abstract
A method of testing an animal for pregnancy or identifying the
sex of the animal comprising the steps of first, providing a first
vessel containing a liquid and having a removable surface wherein
said removable surface is at least partially coated with an
antibody and then introducing a bodily fluid from the female animal
into said first vessel so that said bodily fluid contacts the
liquid and then manipulating the first vessel so that the liquid
contacts the antibody. Then, a second vessel containing a reporter
hormone solution is provided and the removable surface from the
first vessel is displaced to the second vessel and manipulating the
second vessel so that the reporter hormone solution contacts the
removable surface. Then, a third vessel containing an indicating
solution which has an appearance which is related to the amount of
the reporter hormone contacted is provided, and the removable
surface is displaced from the second vessel to the third vessel.
The third vessel is manipulated so that the indicating solution
contacts the removable surface. Then, a determination is made
regarding the pregnancy or sex based on the appearance of the
indicating solution.
Inventors: |
D'Aurora; Vito J.; (Bella
Vista, CA) |
Correspondence
Address: |
SAND & SEBOLT
AEGIS TOWER, SUITE 1100, 4940 MUNSON STREET, NW
CANTON
OH
44718-3615
US
|
Assignee: |
DREAM MAKERS, LLC
North Canton
OH
|
Family ID: |
46150274 |
Appl. No.: |
12/713675 |
Filed: |
February 26, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12174348 |
Jul 16, 2008 |
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12713675 |
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10349715 |
Jan 23, 2003 |
7410807 |
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12174348 |
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09912342 |
Jul 24, 2001 |
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10349715 |
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60220279 |
Jul 24, 2000 |
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Current U.S.
Class: |
436/518 ;
422/400 |
Current CPC
Class: |
Y10S 436/814 20130101;
G01N 33/74 20130101; G01N 33/743 20130101; A61D 17/006 20130101;
Y10S 436/811 20130101 |
Class at
Publication: |
436/518 ;
422/61 |
International
Class: |
G01N 33/543 20060101
G01N033/543; B01L 3/00 20060101 B01L003/00 |
Claims
1. A method for testing a mammal for pregnancy comprising the steps
of: (a) introducing a saliva sample from a mammal to be tested into
a first liquid medium to provide a first liquid mixture; (b)
contacting said first liquid mixture with a solid surface, wherein
the solid surface has antibodies against at least one
pregnancy-indicating hormone attached thereto; (c) contact said
solid surface with a reporter hormone solution; (d) contacting said
solid surface with an indicator solution; and (e) determining the
stage of pregnancy of the mammal based on the appearance of either
the solid surface or the indicating solution.
2. The method of claim 1, wherein said mammal is a horse.
3. The method of claim 2, wherein said antibodies are antibodies
against progesterone.
4. A method of testing an animal for pregnancy or identifying the
sex of the animal comprising the steps of: (a) providing a first
liquid medium, a reporter hormone solution, a solid surface
supporting an antibody and then adding to said first liquid medium
either before or after adding the reporter hormone, a bodily fluid
from said female mammal, wherein the animal is a member of a
species of animal and for said species of animal there is a known
concentration of an indicating hormone present in the bodily fluid
at a complete gestation or between male and female animals, and the
amount of antibody on the solid surface approximately corresponds
to said hormone present at a pregnancy of full term for said
species; (b) after the addition of both the reporter hormone and
the bodily fluid to the first liquid medium, contacting said first
liquid medium, bodily fluid and reporter hormone solution with the
solid surface supporting the antibody; (c) contacting the solid
surface supporting the antibody with an indicating solution; and
(d) then making a determination regarding the pregnancy or sex of
the animal based on the appearance of either the solid surface
supporting the antibody or the indicating solution.
5. The method of claim 4, wherein the indicating hormone is
selected from the group consisting of progesterone and estrogen or
other similar hormones indicative of pregnancy or sex.
6. The method of claim 5, wherein in step (a) the indicating
hormone reacts with at least some of the antibody on the removable
surface.
7. The method of claim 5, wherein in step (b) the reporter hormone
fills any antibody with which the indicating hormone does not
fill.
8. The method of claim 4, wherein the reporter hormone is selected
from the group consisting of any compound that, when reacted with
the indicating hormone, changes color.
9. The method of claim 5, wherein on the completion of step (b) the
occurrence of reporter hormone on the solid surface will be
relatively greater when the occurrence of indicating hormone in the
bodily fluid will be relatively greater.
10. The method of claim 4, wherein in step (c) the appearance which
is related to the amount of the reporter hormone contacted is a
color which becomes more intense depending on the amount of
reporter hormone which is contacted.
11. The method of claim 4, wherein the animal is a female mammal in
the case of the pregnancy test or is a monomorphic animal in the
use of the sex identification test.
12. The method of claim 4, wherein the bodily fluid is added to the
first liquid medium before the addition of the reporter
hormone.
13. The method of claim 4, wherein the bodily fluid is added to the
first liquid medium after the addition of the reporter hormone.
14. A method of testing an animal for pregnancy or identifying the
sex of the animal comprising the steps of: (a) providing a first
vessel containing a liquid and having a removable surface wherein
said removable surface is at least partially coated with an
antibody and then introducing a bodily fluid from the female animal
into said first vessel so that said bodily fluid contacts the
liquid and then manipulating the first vessel so that the liquid
contacts the antibody; (b) providing a second vessel containing a
reporter hormone solution and displacing the removable surface from
the first vessel to the second vessel and manipulating the second
vessel so that the reporter hormone solution contacts the removable
surface; (c) providing a third vessel containing an indicating
solution which either causes a change of appearance in the
removable surface or which undergoes change of appearance itself
related to the amount of reporter hormone on the removable surface
and displacing the removable surface from the second vessel to the
third vessel and manipulating the third vessel so that the
indicating solution contacts the removable surface; and (d) then
making a determination regarding the pregnancy of the animal based
on the appearance of either the removable surface or the indicating
solution.
15. The method of claim 14, wherein after step (b) and before step
(c) there is performed an additional step (e) of providing a fourth
vessel containing a wash solution and displacing the removable
surface from the second vessel to the fourth vessel and then
manipulating the fourth vessel to contact the removable surface
with the wash solution.
16. The method of claim 14, wherein in step (d) a visual comparison
means is provided for assistance in determining the appearance of
the removable surface.
17. The method of claim 14, wherein in step (d) a visual comparison
means is provided for assistance in determining the appearance of
the indicating solution.
18. The method of claim 14, wherein in step (a) the liquid in the
first vessel is a buffer liquid.
19. The method of claim 18, wherein the buffer liquid is selected
from the group consisting of any buffer in which the antibody,
hormone and solid phase are stable.
20. The method of claim 14, wherein the first vessel is a vial.
21. The method of claim 20, wherein the vial has a cap having an
inner surface and the removable surface is said inner surface on
said cap.
22. The method of claim 14, wherein the bodily fluid is introduced
into the first vessel on a swab.
23. The method of claim 14, wherein the bodily fluid is selected
from the group consisting of blood, plasma, urine, milk and
saliva.
24. The method of claim 23, wherein the bodily fluid is saliva.
25. The method of claim 14, wherein the first vessel is manipulated
by inverting said first vessel.
26. The method of claim 14, wherein in step (a) there is an
indicating hormone present in the bodily fluid which is related to
pregnancy, and the amount of antibody on the removable surface is
related to the indicating hormone which would be present at
complete gestation.
27. The method of claim 26, wherein the indicating hormone is
selected from the group consisting of progesterone and estrogen or
other hormones indicative of pregnancy or sex.
28. The method of claim 27, wherein in step (a) the indicating
hormone reacts with at least some of the antibody on the removable
surface.
29. The method of claim 28, wherein in step (b) the reporter
hormone fills any antibody with which the indicating hormone does
not fill.
30. The method of claim 14, wherein the reporter hormone is
selected from the group consisting of any compound that, when
reacted with the indicating hormone, changes color.
31. The method of claim 14, wherein the second vessel is a
vial.
32. The method of claim 26, wherein on the completion of step (b)
the occurrence of reporter hormone on the removable surface will be
relatively greater when the occurrence of indicating hormone in the
bodily fluid will be relatively greater.
33. The method of claim 14, wherein in step (c) the appearance
which is related to the amount of the reporter hormone contacted is
a color which becomes more intense depending on the amount of
reporter hormone which is contacted.
34. The method of claim 15, wherein unwanted materials are
contained in the bodily fluid and the reporter solution and the
wash solution removes such unwanted materials.
35. The method of claim 16, wherein in step (e) the visual
comparison means is a control strip having a color displayed in a
plurality of intensities.
36. The method of claim 14, wherein the animal is a female mammal
in the case of the pregnancy test, or the animal is a sexually
monomorphic animal in the case of the sex identification test.
37. A method of testing an animal for pregnancy or sex
identification comprising the steps of: (a) providing a first vial
containing a buffer liquid and having a removable cap having an
inner surface which is at least partially overlaid with a solid
phase antibody coating, and then introducing a bodily fluid from
the female animal into said first vial so that said bodily fluid
contacts said buffer liquid and then manipulating the first vial so
that the buffer liquid contacts the solid phase antibody coating on
the inner surface of the cap; (b) then providing a second vial
containing a reporter hormone, and removing the cap from the first
vial to said second vial, and then manipulating the second vial so
that the reporter hormone solution contacts the solid phase
antibody coating on the inner surface of the cap; and then (c) then
providing a third vial which has an indicating solution which has
an appearance which is related to the amount of reporter hormone
contacted, and removing the cap from the second vial to said third
vial, and manipulating the third vial so that the indicating
solution contacts the solid phase antibody coating on the inner
surface of the cap; and (d) then making a determination regarding
the pregnancy or sex of the animal based on the appearance of the
indicating solution.
38. The method of claim 37, wherein after step (b) and before step
(c) there is performed an additional step (e) of providing a fourth
vial containing a wash solution and removing the cap from the
second vial to the fourth vial and manipulating the fourth vial to
contact the solid phase antibody coating on the inner surface of
the cap with the wash solution.
39. The method of claim 37, wherein in step (d) a visual comparison
means is provided for assistance in determining the appearance of
the indicating solution.
40. A kit for use in testing an animal for pregnancy or sex
identification comprising: a first vessel containing a liquid
buffer and having an opening and a removable cap adapted to close
said opening, and said cap having an inner surface which is at
least partially overlaid with a solid phase antibody coating; a
second vessel containing a reporter hormone solution and having an
opening which is adapted to being closed by the removable cap; and
a third vessel containing an indicating solution and having an
opening which is adapted to being closed by the removable cap.
41. The kit of claim 40, wherein there is a means for introducing a
bodily fluid into the first vessel.
42. The kit of claim 41, wherein the means for introducing the
bodily fluid into the first vessel is a swab.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation of U.S. patent
application Ser. No. 12/174,348, filed Jul. 16, 2008, which is a
divisional application of U.S. patent application Ser. No.
10/349,715, filed Jan. 23, 2003, now U.S. Pat. No. 7,410,807, which
is a continuation-in-part of U.S. patent application Ser. No.
09/912,342, filed Jul. 24, 2001, which application claims priority
from U.S. Provisional Patent Application Ser. No. 60/220,279, filed
Jul. 24, 2000; the disclosures of which are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Technical Field
[0003] The present invention relates to molecular biology and
microbiology and, more particularly, to pregnancy tests for mammals
and sex determination in animals. Still more particularly, this
invention relates to pregnancy tests for equine mammals which are
based on saliva or other bodily fluids.
[0004] 2. Background Information
[0005] The prior art discloses a number of ways to test for
pregnancy in female mammals. U.S. Patent No. 3,968,011 to Manantou,
et al., for example, discloses a method for colorimetrically
assaying the quantity of N-acetyl-.beta.-glucosaminidase in a
female biological medium, such as saliva, which quantity is indicia
of fertility or pregnancy. The implement is an absorbent material,
such as paper strip, impregnated with a phenolic derivative of
N-acetyl-.beta.-d-glucosamine that reacts in the presence of the
glucosaminidase at an acid pH to form a phenol that has a distinct
color at an alkaline pH, and a buffer that maintains said acid pH.
The method may be carried out by wetting the implement with the
medium, allowing the phenol to form, raising the pH to alkalinity
by wetting the implement with an appropriate buffer solution, and
comparing the color of the implement with a color standard.
[0006] U.S. Pat. No. 4,003,988 to Hoff, et al. discloses a direct
agglutination reagent for pregnancy testing which comprises the use
of suspensions of polystyrene latex particles sensitized with a
globulin fraction of anti-serum to human chorionic gonadotropin
(HCG). When mixed with urine or blood serum samples containing HCG,
this reagent agglutinates indicating a positive test for
pregnancy.
[0007] U.S. Pat. No. 4,123,504 to Banik, et al. discloses a method
and device for detecting pregnancy. This test involves
concentration by ultra-filtration of a sample of urine or serum
from a subject; followed by determining the presence of human
chorionic gonadotropin or of its .beta.-subunit in the concentrated
sample.
[0008] U.S. Pat. No. 4,348,207 to Cappel discloses a method for
testing for pregnancy in humans in which a sample of a patient's
first morning urine is added to a test tube containing a known
lyophilized reagent. The tube is capped, shaken to mix the
contents, and placed upright for one to two hours. The tube is then
inverted and compared with positive and negative standard vials to
show either agglutination of particles, such as red blood cells
contained therein, in which case the subject is not pregnant, or a
failure to agglutinate, in which event the patient is pregnant. The
test tube is of sufficient dimension to support capillary action
and is formed from, or has its interior surface coated with, a
material which is non-wettable to the liquid contained therein.
[0009] U.S. Pat. No. 4,716,123 to Wood discloses a home pregnancy
test in which a specifically binding biomaterial is attached to a
macroextensive surface of a plastic strip or the like. A biological
substance which is a specific binding partner to a binding site of
the specifically binding biomaterial is attached to each of a
plurality of synthetic particles. The particles are of a
pre-selected size, refractive index, or the like to enhance their
visibility in accordance with the Mie scattering phenomenon.
Testing is by either contacting the particles with the strips to
obtain adherence of the particles to the strips, or by exposing
strips having the particles already adhering to them to a solution
containing either the specifically binding biomaterial or the
biological substance, whereby the particles adherence to the strip
is eliminated.
[0010] U.S. Pat. No. 4,720,455 to Babu, et al. discloses a
progesterone concentration level test for mammalian body fluids
particularly adapted for milk whereby estrus and pregnancy can be
determined. The test can be carried out with a kit of several
reagents, test tubes and a dip-stick carrying an anti-progesterone
monoclonal antibody.
[0011] U.S. Pat. No. 5,837,197 to Porrazzo, et al. discloses a
fertility analysis and reproductive health system that is
applicable to both female and male mammals. The invention disclosed
in this patent is a portable, handheld, integrated unit which can
be manufactured out of plastic. The unit can be disposable for
hygienic purposes, or cleaned or sterilized for repeated use as
desired. Aspects of the invention include the following: an
immediate testing methodology employing any and all female fluids
or secretions called positive fertility testing; the creation of a
plastic, completely integrated, portable, self-contained and
self-focusing examination system which relies on a visual reference
system making it language independent; a test area section with
replaceable slides where different, specific wavelengths of light
are employed; the embodiment of a compound test areas so that
multiple positive fertility tests may be conducted simultaneously;
implementing the ability to immediately perform two or more
positive fertility tests simultaneously using different female
fluids or secretions; providing a novel batter powered
microprocessor system to automatically perform the positive
fertility testing; providing an accurate indicator of positive
ovulation whereby the woman may pinpoint times of greatest
fertility, thereby knowing the optimum time period for achieving
pregnancy.
[0012] The prior art also discloses possibly relevant art
concerning methods of detecting ovulation in female mammals. U.S.
Pat. No. 4,385,125 to Preti, et al., for example, discloses that
the level of certain alcohols is dramatically increased during the
ovulation cycle of mammals. This patent thus utilizes this change
to precisely ascertain the time of ovulation by monitoring the
concentration of alcohols noting that a spike in the concentration
indicates a time of ovulation. While this device and process of
this patent determines the alcohol level by measuring such alcohol
levels in saliva, it does not utilize reporter enzymes to measure
the level of certain hormones in saliva to determine pregnancy, but
only to measure alcohol to determine ovulation.
[0013] U.S. Pat. No. 5,460,976 to O'Connor discloses a method of
predicting ovulation and a test kit is described which allows one
to accurately predict the time of ovulation in an animal in advance
thus permitting the highest rate of pregnancy to be achieved and at
the same time minimizing embryonic death.
[0014] U.S. Pat. No. 5,721,142 to Klemm, et al. also relates to a
method of monitoring the mammalian reproductive cycles. In the
method disclosed in this patent the quantity of one or more low
molecular weight compounds such as acetal/dehyde are monitored.
[0015] U.S. Pat. No. 5,837,197 to Porrazzo, et al. discloses a
positive fertility testing and reproductive health system. Inasmuch
as this patent relates generally to the measurement of reproductive
health and fertility status, it discloses a number of the general
features of the invention including testing for enzymes in saliva
to determine a male or female's reproductive state and health.
[0016] U.S. Pat. No. 5,914,271 to Law, et al., discloses a
fertility test and more particularly, relates to the testing of
magnesium and calcium concentrations in saliva. More specifically,
within the three to five days immediately preceding ovulation, the
calcium and magnesium concentrations in the saliva drop. As a
result, concentration monitoring can be done by any conventional
means for quantitatively analyzing the calcium and magnesium to
determine the fertility state of the user.
[0017] A need still exists, however, for improved ways of testing
for pregnancy in mammals.
[0018] Many species of avians as well as other animals also do not
display phenotypic differences between the sexes, especially in
immature individuals. With respect to avians, such sex
identification may be difficult in some commercial species such as
parrots and ratites, and for certain endangered species such as the
California Condor and Whooping Crane as well as non-endangered
species, such as the Canada Goose.
[0019] Gender identification can be performed in sexually
monomorphic birds and other animals through surgical examination.
Alternatively, sex identification can sometimes be made through
karyotype analysis.
[0020] Another alternative method of sex identification in birds is
disclosed in U.S. Pat. No. 5,679,514 to Baker. This method
comprises the steps of obtaining a DNA sequence which includes the
W specific chromosome from the bird. Then, there is the step of
identifying the nucleotides of the DNA sequence of the W specific
chromosome. This prior art method also pertains to a method of
determining the sex of a bird, and a method of determining genetic
relatedness between two birds.
[0021] The above described methods of sex identification may,
however, be relatively expensive and time consuming. A need,
therefore, exists for an improved method of sex identification for
monomorphic avians and other animals.
SUMMARY OF THE INVENTION
[0022] It is an object of the present invention to provide an
accurate, easy and inexpensive means for detecting pregnancy in
equines or other mammals and for identifying sex in monomorphic
avians and other animals. It is another object of the present
invention to provide a test for pregnancy in equines or other
mammals or for sex identification in monomorphic avians and other
animals which can be performed at home and without laboratory
facilities and without specialized veterinary or medical
training.
[0023] It is another object of the present invention to provide a
test for pregnancy in equines and other animals and for sex
identification in monomorphic avians and other animals which is
sensitive enough to allow use of saliva as the bodily fluid.
[0024] It is still another object of the present invention to
provide a test for pregnancy in equines and other animals and for
sex identification in monomorphic avians and other animals which
has a low level of ambiguity and a high level of reliability.
[0025] It is still another object of the present invention to
provide a test for pregnancy in equines and other mammals and for
sex identification in monomorphic avians and other animals which
provides the ability to provide prompt test results to the
user.
[0026] Another object of the present invention is to provide a
simple and easy to use kit by means of which tests for pregnancy in
equines or other mammals or for sex identification in monomorphic
avians and other animals may be performed at home or on a farm or
ranch by individuals without specialized veterinary or medical
training.
[0027] These and other objects are met by the present invention
which is a method of testing a female mammal for pregnancy or
identifying the sex of an animal comprising the steps of first
providing a first liquid medium and then introducing a bodily fluid
from the female animal into said liquid and then providing a solid
surface supporting an antibody and contacting said liquid with the
solid surface supporting the antibody. Then a reporter hormone
solution is provided and the solid surface supporting the antibody
is displaced to place said solid surface supporting the antibody in
contact with the reporter hormone solution. Then the solid surface
supporting the antibody is contacted with an indicating solution. A
determination is then made regarding pregnancy or sex of the animal
based on the appearance of either the solid surface supporting the
antibody or the indicating solution. It is found that the use of
the reporter solution in a separate step makes the test more
sensitive so as to facilitate the use of the test with saliva.
[0028] The present invention also encompasses a method of testing a
female mammal for pregnancy or identifying the sex of animal
comprising the steps first providing a first liquid medium, a
reporter hormone solution, a solid surface supporting an antibody
and then adding to said first liquid medium either before or after
adding the reporter hormone, a bodily fluid from said female
mammal. The female animal is a member of a species of animal and
for that species of animal there is a known concentration of an
indicating hormone present in the bodily fluid that varies during
pregnancy. The amount of antibody on the solid surface is titrated
to give the most sensitivity between the presence and absence of
indicating hormone for said species. After the addition of both the
reporter hormone and the bodily fluid to the first liquid medium,
the first liquid medium is contacted with the solid surface
supporting the antibody. The solid surface supporting the antibody
with various levels of reporting hormone depending on the native
hormone in the bodily fluid, is contacted with an indicating
solution. A determination is then made regarding the pregnancy of
the female animal or sex of the animal based on the level of color
on either the solid surface supporting the antibody or the
indicating solution, depending on the reporting solution used. It
is found that ambiguity of the test is reduced when the amount of
antibody on the solid surface is titrated precisely to the minimum
concentration required yet high enough to produce sufficient
color.
[0029] The present invention also encompasses a method of testing a
female mammal for pregnancy or identifying the sex of an animal
comprising the steps of first, providing a first vessel containing
a liquid and having a removable surface wherein said removable
surface is at least partially coated with an antibody and then
introducing a bodily fluid from the female animal into said first
vessel so that said bodily fluid contacts the liquid and then
manipulating the first vessel so that the liquid contacts the
antibody. Then, a second vessel containing a reporter hormone
solution is provided and the removable surface from the first
vessel is displaced to the second vessel and manipulating the
second vessel so that the reporter hormone solution contacts the
removable surface. Any antibody not containing hormone from the
bodily fluid will then bind the reporter hormone. Then, a third
vessel containing an indicating solution which has an appearance
which is related to the amount of the reporter hormone contacted is
provided, and the removable surface is displaced from the second
vessel to the third vessel. The third vessel is manipulated so that
the indicating solution contacts the removable surface. Then, a
determination is made regarding the pregnancy of the female or sex
of the animal based on the appearance of the indicating solution in
the vessel or intensity of color on the removable surface where the
antibody and reporter hormone are attached.
[0030] The invention also encompasses a method of testing a female
mammal for pregnancy or identifying the sex of an animal comprising
the steps of providing a first vial containing a buffer liquid and
having a removable cap having an inner surface which is at least
partially overlaid with a solid phase antibody coating. A bodily
fluid from the female animal is then introduced into said first
vial so that said bodily fluid contacts said buffer liquid and the
first vial is then manipulated so that the buffer liquid contacts
the solid phase antibody coating on the inner surface of the cap. A
second vial containing a reporter hormone is then provided, and the
cap is removed from the first vial to said second vial, and the
second vial is then manipulated so that the reporter hormone
solution contacts the solid phase antibody coating on the inner
surface of the cap. A third vial is provided which has an
indicating solution which has an appearance which is related to the
amount of reporter hormone contacted. The cap is removed from the
second vial to said third vial, and the third vial is manipulated
so that the indicating solution contacts the solid phase antibody
coating on the inner surface of the cap. A determination regarding
the pregnancy of the female animal or sex of the animal based on
the appearance of the indicating solution in the vial or intensity
of color on the removable cap where the antibody and reporter
hormone are attached.
[0031] The invention also encompasses a kit for use in testing a
female animal for pregnancy or identifying the sex of an animal
which includes a first vial containing a liquid buffer and having
an opening and a removable cap adapted to close said opening and
having an inner surface which is at least partially overlaid with a
solid phase antibody coating. There is also a second vial
containing a reporter hormone solution and having an opening which
is adapted to being closed by the removable cap. Also included in
the kit is a third vial containing an indicating solution and
having an opening which is adapted to being closed by the removable
cap. The number of vials may vary depending on the specific
hormones of interest, the species-specific hormones and their
characteristics, the reporter hormones chosen, or other components
of the test. Each component can influence the necessary steps to
optimize the invention. With respect to the test for determining
the sex of animals, in some animals visual observations are
insufficient to determine whether the animal is male or female. For
example, this test would be able to determine the sex of birds and
reptiles, whose genitalia are not visible without significant
manipulation of the animal. For instance, estrogen and it's
derivatives are found in significantly higher levels in female
animals than in male animals. Other hormones also have a
significant differential concentration between the male and female
members of an animal species. Testing for these hormones would
enable one to determine the sex of the animal. The list of hormones
and animals is prohibitively large for this application, but is
well known from numerous literature references.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] The preferred embodiment of the invention, illustrative of
the best mode in which applicant contemplated applying the
principles, is set forth in the following description and is shown
in the drawings and is particularly and distinctly pointed out and
set forth in the appended claims.
[0033] FIGS. 1a-7 show a kit by means of which the method of the
present invention may be carried out, wherein FIG. 1a is a bottom
plan view of a solid phase antibody cap section of a preferred
embodiment of the kit of the present invention in which the
protective foil cover of this cap is in place;
[0034] FIG. 1b is a cross section through 1b-1b in FIG. 1a;
[0035] FIG. 1c is a bottom plan view of a solid phase antibody cap
section shown in FIG. 1a in which the protective foil cover has
been removed;
[0036] FIG. 1d is a cross section through 1d-1d in FIG. 1c;
[0037] FIG. 2 is a front elevational view of the first vial section
of a preferred embodiment of the kit of the present invention;
[0038] FIG. 3 is a front elevational view of the swab section of a
preferred embodiment of the kit of the present invention;
[0039] FIG. 4 is a front elevational view of the second vial
section with protective foil cover of the kit of the present
invention;
[0040] FIG. 5 is a front elevational view of the indicator strip
section of the kit of the present invention;
[0041] FIG. 6 is a front elevational view of the preferred
embodiment of the third vial section of the kit of the present
invention;
[0042] FIG. 7 is a front elevational view of the fourth vial
section of the kit of the present invention;
[0043] FIGS. 8a, 8b, and 8c are successive perspective views of the
first vial section, the solid phase antibody cap section, and the
swab section in which the first step of a preferred embodiment of
the method of the present invention is illustrated;
[0044] FIGS. 9a, 9b, and 9c are successive perspective views of the
second vial, the solid phase antibody cap, and the strip in which
the second step of the preferred embodiment method of the present
invention is illustrated;
[0045] FIGS. 10a, 10b, and 10c are each perspective views of the
third vial, the solid phase antibody cap, and the indicator strip
in which the third step of the preferred embodiment of the present
invention is illustrated;
[0046] FIGS. 11a, 11b, and 11c are successive perspective views of
the fourth vial, the solid phase antibody cap, and the indicator
strip illustrating fourth step in the preferred embodiment of the
present invention; and
[0047] FIGS. 12a, 12b and 12c are plan views of the vial tap and
indicating strip illustrating fifth step of the present
invention.
[0048] FIG. 13 is a graph showing equine levels of progesterone in
saliva at various stages of pregnancy.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0049] Referring particularly to FIGS. 1a-7, a kit which may be
used in the method of the present invention is shown. This kit
includes a solid phase antibody cap section which is shown in FIGS.
1a-1d generally at numeral 10. This cap has a top 12 and a sidewall
14 having an inner thread 16. At the base of the sidewall there is
a foil protective cover 18 as is shown in FIGS. 1a and 1b which may
be removed as is shown in FIGS. 1c and 1d. On the inner side of the
top 12 there is a solid phase antibody layer 20. This solid phase
antibody layer is a layer or layers of proteins, including the
antibody and possibly other enhancing proteins and include various
antibodies and various other enhancing proteins and processes. The
solid phase may be any material that has a natural irreversible
affinity for proteins. Non-limiting examples include nitrocellulose
paper, diatomaceous earth coated onto a surface, and plastics. The
solid phase material may also be any material that is chemically
reactive to proteins where on proteins can be chemically attached
to the material. Non-limiting examples include plastics, paper
materials, and polymers. The antibodies can be any monoclonal or
polyclonal antibody to any hormone or combination of hormones that
are indicative of pregnancy or whose levels increase or decrease
during pregnancy, and examples of which include antibodies directed
against progesterone, estrogen, follicle stimulating hormone,
leutinizing hormone, other steroid hormones or their derivatives or
metabolites, and other protein hormones or their derivatives or
metabolites. Antibodies may also be any antibody listed above from
any species that cross reacts sufficiently between species to be
used to detect pregnancy in a different species than the
originating species of the antigen. Enhancing proteins and
processes include artificial antibodies generated by various
processes. Non-limiting examples include proteins that are not
antibodies but have similar affinities for any hormones listed
above, and antibodies that are manufactured from parts of multiple
antibodies, such as the combination of more than one antibody
reactive site into a single protein body that functions like an
antibody. Multiple layers of proteins laid down on any surface in
(1a) that are designed to enhance the sensitivity of the system,
reduce non-specific binding, and increase the stability of the
antibody coating. Antibody levels on the solid phase can be
adjusted to increase or decrease the sensitivity of the assay. As
an example, equine levels of progesterone in saliva vary with the
state of pregnancy. Within days of the start of gestation, the
levels of progesterone increase. Measuring the presence of
progesterone in saliva is indicative of pregnancy. Solid phase
antibody consists of an anti-progesterone polyclonal antibody
against progesterone on a nitrocellulose paper (solid phase)
creating a "spot" of antibody. The liquid antibody solution is
applied to a very small area of the solid phase using a small
pipette. To get the proper amount of antibody on the solid phase,
multiple applications can be made. This is done by applying the
antibody solution and letting it dry and repeating the process
until the proper amount of antibody is absorbed onto the solid
phase, without spreading the antibody spot by applying a large
amount of antibody solution in one applications. The amount of
antibody that gives the most sensitive assay is determined by
creating several test solid phase spots with differing amounts of
antibody and testing the solid phase with saliva from mares of
various states of pregnancy to determine that level of antibody
that distinguishes the various states best. The solid phase with
the correct level of antibody in the spot is put into the cap or
strip (depending on the method used to perform the assay), as
described in the figures, and is used for the assay as described.
An alternative to the development of color on the solid phase spot,
the assay can be designed so that a solution generates a color
change that is indicative of the state of pregnancy.
[0050] Another element of the kit of the present invention is a
first vial which is shown in FIG. 2 generally at numeral 22. This
vial is cylindrical in shape, has a sidewall 24. There is a thread
28 on the outside of sidewall 24 for engagement with the thread 16
on the cap 10. The first vial 22 has a temporary foil protective
cover 30 which may be removed at the time of the test as is
described hereafter to provide an open top 32. Inside the first
vial 22 there is a liquid buffer solution 34 which may be any
buffer in which the antibody, hormone, and solid phase are stable.
The buffer will contain an antimicrobial component to inhibit
spoilage. The buffer may also contain a surfactant or detergent
that could help to minimize non-specific binding of assay
components to any physical surface. Non-limiting examples include
buffering components such as TRISMA buffer with a pH near 7; or
phosphate buffered saline, consisting of various ortho phosphate
salts in a sodium chloride saline solution to achieve a pH near 7.
The buffer may also be antimicrobial such as sodium azide and
thimerosol. The buffer may also be detergent/surfactants such as
TWEEN-20, TWEEN-80, or sodium dodecylsulfate. Buffering components,
antimicrobials and detergents/surfactants are chosen based on the
other components of the assay kit. As will be appreciated by those
skilled in the art, certain reporter enzymes, chromophores, and
hormones are more stable in certain combinations of buffer
components.
[0051] The kit of the present invention also includes a swab which
is shown in FIG. 3 generally at numeral 36. This swab 36 includes a
stick 38 with a break point 39, a handle 40 and an absorbent cotton
section 41. Also included in the kit of the present invention is a
second vial shown generally in numeral 42 in FIG. 4 which is
essentially identical in structure to vial 22 except that it
contains a reporter hormone solution 44 rather than the buffer
solution 34. The kit of the present invention also includes a strip
45 which is shown in FIG. 5. The strip 45 has a grip end 46 and a
color end 47. The function and operation of this strip is described
hereafter. The kit also includes a third vial 48 which is
essentially to vial 24 except that it contains a liquid wash
solution 50 rather than the buffer solution 34. Finally, the kit
includes a fourth vial shown generally at numeral 52 in FIG. 7
which is also essentially identical to the first vial 22 except
that it contains a reporter solution 54 instead of the buffer
solution 34.
[0052] The reporter solution is a compound that, when reacted with
the reporter hormone/enzyme, changes color. The assay can be
optimized so that the color can either be seen on the spot on the
cap or in the vial solution. It is one of the substrates of the
enzyme portion of the reporter hormone complex. Non-limiting
examples include 3,3',5,5'-tetramethylbenzidine for use with
horseradish peroxidase reporter enzyme; p-nitrophenylphosphate with
alkaline phosphatase reporter and -nitrophenyl-D-galactopyranoside
(ONPG) with galctosidase.
[0053] Referring to FIGS. 8a-11c, the method of this invention is
illustrated. The first step in this method is illustrated with
particular reference to FIGS. 8a-8c. In FIG. 8a the first vial or
vessel 22 with the foil protective cover 30 removed is collected
with the cap 10 and the swab 36. The swab is then saturated with
saliva from a mare and is then inserted through the open top 32
into the buffer solution 34 in the first vial 22. Referring
particularly to FIG. 8b, the cap 10 is then positioned over the
open end 32 of the first vial 22 and attached thereto by the mating
screw threads 16 and 28. Referring to FIG. 8c, the first vial 22 is
then inverted so that the buffer solution 34 and the inner solid
phase antibody layer 20 are in contact.
[0054] It is in this step that the equine hormone binds to the
antibodies. After a fixed time the first vial 22 is stood upright
and the cap with the tightly bound hormones are removed. In order
for this test to be sensitive to the amount of hormone present in
the saliva so as to represent various stages of gestation or the
sex of the animal, the amount of antibody that is chemically
attached to the cap will have been titrated empirically for each
antibody, reporter hormone, and species. Therefore, after step one,
the amount of antibodies that are not bound up with hormone will be
the greatest for those mares that are most removed from complete
gestation.
[0055] Referring to FIGS. 9a-9c, the second step in the method is
shown. Referring particularly to FIG. 9a, the cap 10 is removed
adjacent to the second vial or vessel 42 which contains the
reporter hormone solution 44. The cap 10 is then emplaced on the
second vial 42 as is shown in FIG. 9b. The second vial 42 is then
inverted so that the reporter hormone solution 44 is in contact
with the solid phase antibody layer 20 on the inner side of the top
section of the cap 10. The reporter hormone fills up any remaining
antibodies left from the first step. The number of reporter
hormones, therefore, on the cap at the end of this step is greatest
for saliva with the least hormone, and is the least for saliva with
the maximum hormones.
[0056] Referring to FIGS. 10a-10c, the third step in the method of
this invention is illustrated. Referring particularly to FIG. 10a,
the cap 10 is moved adjacent the third vial or vessel 48 which is
partially filled with the washing solution 50. As is shown in FIG.
10b, the cap 10 is then emplaced on the third vial 48. As is shown
in FIG. 10c, the third vial 48 is then inverted to place the solid
phase antibody layer 20 on the inner side of top 12 of cap 10 in
contact with the wash solution 50 to remove any unwanted materials
from the solid phase antibody layer 20 that may have originated
from the saliva or the reporter solution 44.
[0057] Referring to FIGS. 11a-11c, the cap 10 is removed from the
third vial 48 to a position adjacent the fourth vial or vessel 52
which contains color solution 54. The cap 10 is then inserted onto
the fourth vial 52, and the fourth vial 52 is inverted to place the
color solution 54 into contact with the solid phase antibody layer
20. The intensity of the color of the color solution 54 will depend
on the amount of reporter hormone that was bound to the solid phase
antibody layer 20 while the cap 10 was attached to the second vial
42 in the second step of this method.
[0058] In the final step of the procedure, the color solution 54 in
the fourth vial 52 is compared with a color control strip or other
indicator of a positive hormone presence such as strip 45. The
amount of color is proportional to the amount of reporter hormone
which is dependent on the amount of hormone in the original saliva
sample. The intensity of the color solution 54 in fourth vial 52 is
therefore, an indicator of the gestational state or the sex of the
animal.
EXAMPLE 1
[0059] A kit is prepared as described according to the foregoing.
Supplies and chemicals include a 0.1 M citrate buffer, pH 5.0 with
preservative, anti-progesterone antibody in a neutral buffer
solution, nitrocellulose paper, kit components as described in the
figures, color solution with o-phenylenediamine with 0.012%
H.sub.2O.sub.2, detergent/surfactant as described above which is a
commercially available reporter hormone for example, a commercially
available progesterone conjugated to horseradish peroxidase.
1. Preparation of the Active Removable Surface (Cap)
[0060] The active surface is commercially available nitrocellulose
paper (NCP). NCP irreversibly binds proteins with high affinity.
Antibody is applied to the NCP as described below in (2). NCP is
cut to fit inside the Cap and is attached, possibly with adhesive,
a snap in ring, or other means that do not interfere with observing
the antibody spot when color occurs. The resulting cap is the
Active Cap.
2. Preparation of the Antibody Spot On the NCP
[0061] A commercially available antibody to progesterone is diluted
to a predetermined level wherein multiple applications of
sub-microliter volumes can be applied to the NCP to get a sharp,
small spot for ultimate viewing of the color development. The
antibody is applied to the NCP, let the spot dry, and re-apply the
antibody. This procedure is repeated until the pre-determined
amount of antibody is absorbed to the NCP. The correct amount of
antibody attached to NCP will be that amount that gives the best
differential between saliva of mares that are pregnant or not. This
is determined experimentally using multiple saliva samples from
different pregnant mares and mares who are not pregnant (or male
horses) and empirical observations of hormone binding and reporter
hormone binding and maximum sensitivity.
3. Preparation of Reporter Hormone Solution
[0062] Progesterone-horseradish peroxidase reporter hormone can be
purchased or manufactured. The reporter is diluted in citrate
buffer. The dilution factor depends on the initial concentration of
reporter and is determined experimentally by determining the amount
of reporter that gives the maximum distinction between pregnant and
non-pregnant horses with a specific lot of antibody spot on NCP.
Minimum concentrations that give adequate signal are preferred.
4. Preparation of the Reporter Solution
[0063] approximately 34 mg of o-phenylenediamine is dissolved in
100 ml of 0.1 M citrate buffer at pH 5.0, containing 0.012%
H2O.sub.2. Preservatives and surfactants can be added to a final
concentration of less than approximately 0.1% to inhibit
non-specific binding of either the colorless reporter or its
colored reacted species.
5. Buffer
[0064] The buffer for this assay will be Tris buffer, pH 7.4 with
0.1% TWEEN 20 and 0.1% bovine serum albumin as a stabilizer and is
used in all steps except the final color reaction.
6. Preparing the Kit
[0065] The vials are standard 40 ml screw cap glass vials with
TEFLON-lined caps.
There are four separate vials that will be used during the assay
and are called Vial 1, Vial 2, Vial 3, and vial 4 based on the
sequence in which they are used. Each vial has it's own cap. Only
Vial 1 has an active cap and it will be transferred to each of the
vials as the assay proceeds. As the Vial 1 cap is removed from Vial
1 and put on Vial 2, the cap from Vial 2 will be put on Vial 1 for
safe disposal of Vial 1. As Vial 1 cap is removed from Vial 2 and
put onto Vial 3 during the assay described below, the cap from Vial
3 will be put on Vial 2 for safe disposal of Vial
7. This Procedure Continues For All Four Vials
[0066] Vial 1 contains 30 ml buffer. Vial 2 contains 30 ml of
Reporter Hormone in buffer. Vial 3 contained 40 ml of buffer. Vial
4 contains 30 ml of Reporter Solution.
The pregnancy of the mare is determined as follows:
[0067] The approximately 10 inch cotton swab is put into the mare's
mouth and run around various parts of the mouth until the cotton is
thoroughly moistened with saliva. The swab is then scored (or it
can be scored in advance or purchased scored) at approximately 1
inch length so that the portion containing the cotton can be broken
off and placed into vial 1 and the lid tightened. Vial 1 is
inverted, the cotton should still be submersed in the buffer, and
the cap on vial 1 is in contact with the buffer. These components
are incubated for approximately 30 minutes wherein the mare's
hormones in the saliva bind to the antibody on the Active Cap.
[0068] The active cap is removed from vial 1 and put onto vial 2
containing the reported hormone solution. Vial 2 is inverted to
contact the active cap with the reporter hormone solution. These
components are incubated for approximately 30 minutes. All
remaining antibody binding sites are bound to the reporter
hormone.
[0069] The active cap is removed from vial 2 and put onto vial 3
containing the buffer used as a wash solution to remove excess
reporter hormone. Vial 3 is inverted to contact the active cap with
the buffer. The vial can be gently shaken to facilitate the washing
process. These components are incubated for approximately 30
minutes. This substrate will precipitate on the Active Cap during
the formation of the colored enzyme product. By using alternate
commercially available substrates as indicator solutions, the
colored product of the enzyme reaction can be found in the solution
instead of the Active Cap, and the indicator of pregnancy is the
intensity of the color of the solution in Vial 4. This can be
compared to a supplied color strip where the pregnancy cut-off
level is indicated.
[0070] The active cap is removed from vial 3 and put onto vial 4
containing the reporter solution. Vial 4 is inverted to contact the
active cap with the reporter solution. These components are
incubated for approximately 30 minutes.
[0071] The active cap is removed from vial 4. The color spot on the
active cap is compared to the color strip provided. If the color
spot is lighter than the "pregnant" indicator on the color strip,
then the mare is pregnant.
EXAMPLE 2
[0072] Example 1 can be performed on a semi-rigid material with
properties similar to the active cap. The semi-rigid material can
be manufactured in the shape of a strip or stick. This active strip
will have the anti-progesterone antibody coated on it. Instead of
have an active cap for color development, the active strip will be
the indicator or pregnancy and will be moved from Vial 1 to Vial 4,
instead of the Active Cap in Example 1. In this example, the steps
occur as in Example 1 except that the vials need not be inverted.
As in Example 1, alternate color production schemes may be used
that would result in color development in solution instead of on
the active surface of the strip or stick.
[0073] Beside progesterone as an equine pregnancy indicator, there
are other hormone indicators for equine species. In addition, all
mammals have small peptide hormones that change on the state of
pregnancy. Those skilled in the art will appreciate that it will be
possible to make use of such alternate hormones in the practice of
the method and device of this invention.
[0074] It will also be appreciated by those skilled in the art that
the method and apparatus described herein may be adapted for
identification of the sex of various avians and other animals
without readily identifiable sex characteristics.
[0075] It will be appreciated that a method for detecting
pregnancies in mammals and for identifying sex in monomorphic
avians and other animals and a kit for use therein, has been
described, which method is easy to do, is inexpensive, and may be
performed at home without expensive laboratory facilities or
specialized training. This method and kit also eliminate any
complicated measuring or manipulation requirements for the
user.
[0076] Accordingly, the improved PREGNANCY AND SEX IDENTIFICATION
TEST BASED ON SALIVA OR OTHER BODILY FLUIDS method apparatus is
simplified, provides an effective, safe, inexpensive, and efficient
method and device which achieves the enumerated objectives,
provides for eliminating difficulties encountered with prior
methods and devices, and solves problems and obtains new results in
the art. In the foregoing description, certain terms have been used
for brevity, clearness, and understanding; but no unnecessary
limitations are to be implied therefrom beyond the requirement of
the prior art, because such terms are used for descriptive purposes
and are intended to be broadly construed.
Moreover, the description and illustration of the invention is by
way of example, and the scope of the invention is not limited to
the exact details shown or described.
[0077] Having now described the features, discoveries, and
principles of the invention, the manner in which the PREGNANCY AND
SEX IDENTIFICATION TEST BASED ON SALIVA OR OTHER BODILY FLUIDS is
constructed and used, the characteristics of the construction, and
the advantageous new and useful results obtained; the new and
useful structures, devices, elements, arrangements, parts, and
combinations are set forth in the appended claims.
* * * * *