U.S. patent number 3,875,013 [Application Number 05/333,029] was granted by the patent office on 1975-04-01 for article for detecting the fertile period and method for using same.
This patent grant is currently assigned to Alza Corporation. Invention is credited to Adolfo Rosado Garcia, Jorge Martinez Manautou.
United States Patent |
3,875,013 |
Manautou , et al. |
April 1, 1975 |
Article for detecting the fertile period and method for using
same
Abstract
An article of manufacture and a test is provided for detecting
the fertile period or the presence of pregnancy in the female. The
article is comprised of a porous support impregnated with at least
one color developing compound such as
p-nitrophenyl-n-acetyl-.beta.-d-glucosaminide and at least one
buffer that develops a color when contacted with a female
biological medium. The development of the marked color is
coincident with the fertile period, which indication of the fertile
period can be used either for achieving fertilization or for
preventing conception by the rhythmic method. Also, in the days
preceding the expected menstrual period, the development of the
marked color is coincident with the presence of pregnancy.
Inventors: |
Manautou; Jorge Martinez
(Mexico City, MX), Garcia; Adolfo Rosado (Mexico
City, MX) |
Assignee: |
Alza Corporation (Palo Alto,
CA)
|
Family
ID: |
23300953 |
Appl.
No.: |
05/333,029 |
Filed: |
February 16, 1973 |
Current U.S.
Class: |
435/18; 435/200;
435/806 |
Current CPC
Class: |
C12Q
1/34 (20130101); Y10S 435/806 (20130101); G01N
2333/924 (20130101); C12Q 2334/10 (20130101) |
Current International
Class: |
C12Q
1/34 (20060101); G01n 031/14 () |
Field of
Search: |
;195/13.5R,13.5C |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Tanenholtz; Alvin E.
Attorney, Agent or Firm: Sabatine; Paul L. Mandell; Edward
L. Benz; William H.
Claims
What is claimed is:
1. A test article useful for detecting an increase of
N-acetyl-.beta.-glucosaminadase in a female biological media which
is coincident with the onset of the fertile period of a female,
which article is comprised of a bibulous material containing
impregnated therein a non-toxic buffer capable of maintaining the
pH in the range of 4 to 5 and an effective amount of the color
indicator p-nitrophenyl-n-acetyl-.beta.-d-glucosaminide which in
the presence of an increase of N-acetyl-.beta.-glucosaminadase in
the female biological media forms a color developing compound
useful for detecting the fertile period.
2. A test article useful for detecting an increased
N-acetyl-.beta.-glucosaminadase activity according to claim 1 which
additionally includes as a color intensifier a separate buffer
capable of maintaining the pH in the range of 9 to 11.
3. A test article useful for detecting an increase of
N-acetyl-.beta.-glucosaminadase in a female biological media which
is coincident with the presence of pregnancy in a female, which
article is comprised of a bibulous absorbent strip-shaped material
containing impregnated therein a non-toxic buffer capable of
maintaining the pH in the range of 4 to 5 and an effective amount
of a color indicator selected from the group of indicators
consisting essentially of
p-nitrophenyl-n-acetyl-.beta.-d-glucosaminide,
.alpha.-naphthol-n-acetyl-.beta.-d-glucosaminide,
1-[p-nitrocatechol]-n-acetyl-.beta.-d-glucosaminide and
3,3-bis(p-hydroxyphenyl)phthalid-n-acetyl-.beta.-d-glucosaminide
which indicator in the presence of an increase of
N-acetyl-.beta.-glucosamindase in the female biological media forms
a color developing compound wherein the color developed indicates
the presence of pregnancy.
4. A diagnostic test for the detection of the fertile period of a
female warm-blooded animal which test is comprised of adding a
female media selected from the group consisting of saliva, cervical
fluid and vaginal fluid to a composition consisting of the color
indicator p-nitrophenyl-n-acetyl-.beta.-d-glucosaminide in an
amount sufficient to develop a color in the presence of
N-acetyl-.beta.-glucosaminadase in the media indicating fertility
and ovulation and a biologically acceptable first buffer capable of
maintaining the pH in the range of 4 to 5 to form a color
developing compound, then adding a second buffer capable of
maintaining the pH in the range of 9 to 11 to further develope the
color which indicates the presence of said fertile period of the
female.
5. A method for indicating when to use rhythmic contraception
adapted to a female which method is comprised of determining the
fertile period of the female by adding a female media selected from
the group consisting of saliva, cervical mucosa and vaginal mucosa
to a mixture consisting of the color indicator
p-nitrophenyl-n-acetyl-.beta.-d-glucosaminide present in an amount
sufficient to develop a color in the presence of
N-acetyl-.beta.-glucosaminadase in the media and a biological
acceptable buffer capable of maintaining the pH in the range of 4
to 5 to form a color developing compound, then adding a buffer
capable of maintaining the pH in the range of 9 to 11 to further
develop the color, which color indicates the fertile period that
occurs during ovulation of the female which developed color is a
means for determining when to use said method of rhythmic
contraception.
6. A method for indicating the fertile period of the female that
occurs during ovulation by detecting an increase in the enzyme
N-acetyl-.beta.-glucosamindase which is coincident with the fertile
period of the female wherein the method is comprised of contacting
female saliva containing the increase of
N-acetyl-.beta.-glucosamindase with the color indicator substrate
p-nitrophenyl-n-acetyl-.beta.-d-glucosaminide which is present in
an effective amount to form the color developing compound
p-nitrophenyl in the presence of a buffer capable of maintaining
the pH in the range of 4 to 5, then adding another buffer capable
of maintaining the pH in the range of 9 to 11 to further develop
the color which color indicates the fertile period of the
female.
7. A method for indicating the presence of pregnancy in a female by
detecting an increase in the enzyme N-acetyl-.beta.-glucosaminidase
which is coincident with pregnancy, wherein the method is comprised
of contacting a female fluid excretion with a composition
containing the color indicator substrate
p-nitrophenyl-n-acetyl-.beta.-d-glucosaminide which is contained in
the composition in an effective amount to form the color developing
compound p-nitrophenyl in the presence of a buffer capable of
maintaining the pH in the range of 4 to 5, then adding a buffer
capable of maintaining the pH in the range of 9 to 11 to further
develop the color which color indicates the presence of pregnancy
in the female.
8. A test article useful for detecting an increase of
N-acetyl-.beta.-glucosaminadase in a female biological media which
increase is coincident with the onset of the fertile period of a
female, which article is comprised of a bibulous material
containing impregnated therein a non-toxic buffer capable of
maintaining the pH in the range of 4 to 5 and an effective amount
of the color indicator
.alpha.-naphthol-n-acetyl-.beta.-d-glucosaminide which in the
presence of an increase of N-acetyl-.beta.-glucosaminadase in the
female biological media forms a color developing compound useful
for detecting the fertile period.
9. A test article useful for detecting an increase of
N-acetyl-.beta.-glucosaminadase in a female biological media which
increase is coincident with the onset of the fertile period of a
female, which article is comprised of a bibulous material
containing impregnated therein a non-toxic buffer capable of
maintaining the pH in the range of 4 to 5 and an effective amount
of the color indicator
1-[p-nitrocatechol]-n-acetyl-.beta.-d-glucosaminide which in the
presence of an increase of N-acetyl-.beta.-glucosaminadase in the
female biological media forms a color developing compound useful
for detecting the fertile period.
10. A test article useful for detecting an increase of
N-acetyl-.beta.-glucosaminadase in a female biological media which
increase is coincident with the onset of the fertile period of a
female, which article is comprised of a bibulous material
containing impregnated therein a non-toxic buffer capable of
maintaining the pH in the range of 4 to 5 and the color indicator
3,3-bis(p-hydroxyphenyl)-phthalid-n-acetyl-.beta.-d-glucosaminide
in an effective amount which in the presence of an increase of
N-acetyl-.beta.-glucosaminadase in the female biological media
forms a color developing compound useful for detecting the fertile
period.
Description
BACKGROUND OF THE DISCLOSURE
This invention relates to both a novel article useful for detecting
the fertile period of the female, the presence of pregnancy and to
a novel test method suitable for detecting the said fertile period
or the presence of pregnancy.
In the past, as well as in the present, there has been considerable
interest in detecting the fertile period of the female, that is,
the time during which the female is capable of reproduction, and
also considerable interest in ascertaining the presence of
pregnancy. This interest in detecting the fertile period for having
an offspring, and also to those who wish to abstain during this
period to avoid having an offspring. The interest in detecting the
presence of pregnancy is important to those who desire to provide
medical care for the health of the female and the offspring and
also for interrupting an unwanted pregnancy. However, before this
invention, the means and methods used for ascertaining the fertile
period or the presence of pregnancy have been fraught with
difficulty and uncertainty. For example, one attempt to ascertain
the fertile period or the like was based on the idea that the
fertile period is usually a certain number of days before
menstruation. That is, the period of fertility, or ovulation in the
human female is set from 12 to 16 days before the next menstrual
period and this calculation is used for the intended purpose.
However, calculations based on the onset of menses are often
inaccurate because the human female may ovulate at varying and
unpredictable times. Additionally, the length of life of the ova
and the spermatozoa, which information is not truly known in vivo,
must also be added to the calculated period which further decreases
the value and reliability of this method. Another method often used
for detecting the female period consists in taking basal
temperature, but this method is not only inconvenient, it
additionally requires considerable skill in observing, recording
and interpreting the data. Recently, chemical methods for detecting
the fertile period based on an increased monoesterase or alkaline
phosphatase activity were made available to the art. But, while
these methods represent an advance in the art, they have the
shortcoming of failing to define the fertile period by spreading
their results over a prolonged period, or they failed to detect
pregnancy. Additionally, other prior art methods had the added
shortcoming of lacking a positive ability to effectively ascertain
in a seemingly unequavical manner the presence of pregnancy.
SUMMARY OF THE DISCLOSURE
Accordingly, it is an immediate object of the present invention to
provide an article for detecting the fertile period, or the
presence of pregnancy that substantially overcomes the difficulties
associated with the prior art.
Yet a further object of the present invention is to provide a test
for detecting the fertile period or the presence of pregnancy which
test is convenient, safe, reliable and easy to use.
It is another object of the present invention to provide a simply
chemical fertility or pregnancy test which can be carried out by
the unskilled layman and which test gives results with accuracies
generally better than those obtainable with heretobefore known
fertility tests.
Yet another primary object of the present invention is to provide a
purely chemical test for ascertaining the period of ovulation,
which test does not require animals or complicated calculations,
and which test can be simply accomplished by mixing chemicals with
a female biological medium or contacting the medium with a test
strip that develops a color to indicate the period of
ovulation.
Yet still a further object of the invention is to provide a method
for detecting ovulation which detection can be used as a time for
achieving conception or as a time for rhythmic contraception.
Yet still another object of the invention is to provide a method
for the diagnosis of pregnancy as early as 10 days after
fertilization by the color produced, which method introduces a
considerable advance over the art previously described.
Still a further object of the invention is to provide a method for
earliest detection of the pregnant state which detection can be
used for achieving better medical care.
These and other objects, advantages and features of the present
invention will be more apparent from a further reading of the
specification and the accompanying claims.
DESCRIPTION OF THE INVENTION
Turning now in more detail to the actual and useful test, wherein
the test is comprised of assaying a biological female medium such
as saliva, vaginal fluid, cervical fluid and the like by mixing an
aliquot of the female fluid medium with a color forming reagent and
then observing or measuring the developed color. In one procedure,
the medium and the reagent are mixed in the presence of a first
buffer, and after a short reaction period of about 1 hour, a second
buffer is added to more fully intensify the color and the
absorbance measured in a standard spectrophotometer. In another
test embodiment, a bibulous material is impregnated with a color
developing reagent and a buffer and then contacted with a female
fluid. After a like short period of time, the test material is
dipped or sprayed with a different buffer to more fully develop a
visible color. This color test material then is compared against a
previously prepared color chart. In both embodiments, the color
indicates the fertile period or the presence of pregnancy, and this
knowledge can be used for achieving conception or for rhythmic
contraception or for ascertaining the pregnant state.
The color developing reagent suitable for the purpose of the
present invention is selected from the commercially available
compounds p-nitrophenyl-n-acetyl-.beta.-d-glucosaminide,
.alpha.-naphthol-n-acetyl-.beta.-d-glucosaminide,1-[p-nitrocatechol]-n-ace
tyl-.beta.-d-glucosaminide,
2-[p-nitrocatechol]-n-acetyl-.beta.-d-glucosaminide, and
3,3-bis(p-hydroxyphenyl) phthalid-n-acetyl-.beta.-d-glucosaminide.
Presently, there is at least one of the above color developing
reagents present for the test purpose, although a mixture of these
developers can be used.
The buffers used for the purpose of the invention are of two kinds.
A first buffer system with a pH range of about 4 to 5, and a second
buffer system with a pH range of about 9 to 11. Materials suitable
for compounding the first buffer are sodium citrate and citric
acid, and the buffer used is 0.1 M adjusted to 10.3.
In one embodiment, the test is conveniently carried out by first
mixing a sample of a female fluid, such as, 0.1 ml of saliva with
0.1 ml of buffered substrate comprised of, for example, 0.1 M
sodium citrate-citric acid buffer with 0.2 ml of 0.1 M sodium
citrate buffer. Then, after shaking until the reagents are mixed,
the reagents are permitted to incubate for 30 minutes at room
temperature, to allow the enzyme N-acetyl-.beta.-glucosaminidase
present in the saliva to hydrolytically act on the substrate
p-nitrophenyl-n-acetyl-.beta.-d-glucosaminide. At the end of the
incubation period, a second buffer consisting of 1.0 ml of 1.0 M
sodium glycinate buffer is added to the reaction to stop the
hydrolytic activity and also to develop the color. The absorbance
is measured at 400 nm and compared to a standard curve prepared
with solutions of known concentrations of p-nitrophenol.
Turning now to the embodiment wherein a bibulous material is
impregnated with reagents, generally the bibulous materials
suitable for the practice of the invention are those materials,
which by means of capillary action or any other physical chemical
technique are able to hold liquids. Such materials include test
paper that is preferably an absorbant paper, filter paper,
cellulose strips, wood strips, felt, porous ceramic strips,
assorted cotton substrates and the like. A typical test paper is
prepared for oral use or for contacting cervical or vaginal mucous
by impregnating the porous strip with, for example, 0.1 to 0.5 ml
of buffered substrate consisting of 0.1 M p-nitrophenyl-n acetyl
-.beta.-d-glucosaminide described in 0.1 M sodium citrate buffer.
Then, the porous paper is subjected to drying at room temperature,
or up to 100.degree.C without charing the paper, to evaporate the
aqueous solvent and leave active ingredients on the paper.
The test paper so prepared is used to determine the fertility
period as evidenced by changes in, for example, the saliva of the
female. In the oral test, the female simply touches the test paper
to her tongue to wet it and then waits about 20 to 40 minutes,
usually 30 minutes, at room temperature for the increase in
N-acetyl-.beta.-d-glucosaminide to form a color developing
compound. The color is more fully developed by wetting the test
strip with a small but effective amount of previously prepared 0.1
M sodium glycinate buffer. The test strip is then compared to a
standard color card that has a series of color spots similarly
developed from known concentrations of the color developing
compound p-nitrophenol. The test implement will change or develop a
maximum color during the period of ovulation and fertility of the
female, and it will undergo a minimum or no color change at other
times. This detection can be used for conception or for rhythmic
contraception. The test is used in like fashion for indicating the
presence of pregnancy.
The unobvious and improved results obtained with the test of this
invention are more easily seen in the accompanying Figures. In FIG.
1 there is seen the increased activity of
N-acetyl-.beta.-glucosamindase in saliva during the female
menstrual cycle. The results have been normalized by ascribing a
value of 100 for the highest enzymatic activity during the
menstrual cycle. This value always coincides with the peak basal
body temperature registered at ovulation. In FIG. 1, the arrow
through zero "0" is the beginning of menstruation.
In FIG. 2, the activity of alkaline phosphatase in saliva during
the menstrual cycle is presented as measured by a standard prior
art method to illustrate the improved and unobvious results
obtained by the N-acetyl-.beta.-glucosamindase method of the
invention as seen in FIG. 1. The standard alkaline phosphatase test
is performed by first preparing a sodium or potassium glycinate 0.1
M buffer adjusted to pH 9.6 and a buffered substrate comprised of
0.01 M p-nitrophenylphosphate dissolved in glycine buffer, 0.1 M,
pH 9.6. The test is performed by adding 1.0 ml of buffered
substrate mixture to 0.2 ml of saliva, with shaking and incubation
for 45 minutes at 37.degree.C. After this period of time, the color
developed is measured at 400 nm, nanometer, and compared to a
standard curve. The results in FIG. 2 are presented in a manner
identical with the results presented in FIG. 1.
In FIG. 3, the activity of acid phosphatase in saliva during the
menstrual cycle is presented as measured by a standard prior art
method. This result is presented to illustrate the unobvious and
improved results obtained by the inventive glycosidase method as
seen in FIG. 1. The standard test is carried out by first preparing
an acetic acid sodium acetate 1.0 M buffer adjusted to pH 4.7, a
buffered substrate comprised of p-nitrophenyl-phosphate disodium
salt, 0.1 M, dissolved in 0.1 M sodium acetate buffer adjusted to
pH 4.7, and lastly an 0.1 M sodium alkaline glycine buffer adjusted
to pH 10.3. The test consists of adding 0.1 ml of the buffered
substrate and 0.2 ml of acetate buffer to 0.1 ml of saliva,
followed by shaking and incubation for 30 minutes at room
temperature. At the end of this period, 1.0 ml of glycine buffer is
added to the reaction mixture, with the results measure at 400 nm
and compared to a standard curve.
FIG. 4 is the determination of the peak, second of maximum activity
of N-acetyl-.beta.-glucosamindase and its relationship with
pregnancy. In FIG. 4, the graft is normalized so that the first day
of menstruation occurred or was expected to occur. The vertical
lined area of the curved area is the range of 40 female patients
with normal menstruation. The horizontal lined area of the curved
area is the range of pregnancy of eight patients who used the test
for achieving this result. The numbers on the left of the Figure
represent the optical density, that is, the absorbance or intensity
of the color developed for the test.
In summary, the present invention relates to an improved test, an
article and method which are especially useful for determining the
female fertile period or the presence of prenancy. The test
comprises a color developing compound and buffers, and the test
article is a bibulous material impregnated with same; and, while
the invention is described in detail, it is understood that the
foregoing detailed description is given for the purpose of
illustration and that many variations may be made therein without
departing from the spirit of the invention.
* * * * *