EP0030560A1 - Indicateur specifique d'ovulation imminente - Google Patents
Indicateur specifique d'ovulation imminenteInfo
- Publication number
- EP0030560A1 EP0030560A1 EP80901384A EP80901384A EP0030560A1 EP 0030560 A1 EP0030560 A1 EP 0030560A1 EP 80901384 A EP80901384 A EP 80901384A EP 80901384 A EP80901384 A EP 80901384A EP 0030560 A1 EP0030560 A1 EP 0030560A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- peroxidase
- estrogen
- induced
- solid support
- substrate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/743—Steroid hormones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to methods of detecting im- pending ovulation in human females, including methods which are sufficiently simple so that a woman can carry out the test on herself without the aid of a physician.
- thermometry the basal body temperature method
- ovulation detection methods involving an examination of cervical mucus for its flow properties, saline content, glucose content and the like, are also deficient in that they do not easily lend themselves to self-examination by the woman and require sampling of the posterior portion of the vagina.
- microscopic examination of vaginal cells for staining characteristics and morphology require expensive apparatus and involve techniques which are usually beyond the skill of the average woman. Estrogen analysis of blood and urine is likewise complicated and difficult to carry out.
- EIP estrogen-induced peroxi- dase
- An ElP-antibody combination possesses peroxi ⁇ dase activity, hence a positive peroxidase test on the aforesaid bibulous mat which had been in contact with a vaginal fluid sample and washed, indicates that EIP was present in the vaginal fluid. A negative test for peroxi ⁇ dase indicates the absence of EIP. If EIP is found, it indicates impending ovulation.
- An advantage.of the pre ⁇ sent invention is that a specific antibody to EIP is used and this antibody combines only with EIP and not with the interfering s bstances. Chromogenic substrates of peroxidase and a hydroperoxide are used as a test for peroxidase.
- a simple method for the detection of impending ovulation in women, as in the present invention, is of the highest importance to the human race and is of para- mount interest to such prestigious organizations as the Population Council, the Ford Foundation, the National Institutes of Health and the World Health Organization.
- the present invention a woman can, by herself, de ⁇ termine her impending fertile time.
- the fertile time com ⁇ mences within 12 to 72 hours of ovulation (C. Tietze Fertility and Sterility, Vol. 11, p. 485, 1960).
- this form of birth control could, if practiced widely, substantially reduce the rate of world population growth.
- a woman practicing abstinence during the fertile period as determined by the present invention may avoid the need for contraceptive devices, such as the intrauterine device, for birth control which are considered undesirable to some.
- the present invention may also be an aid to couples who wish to have a child, but have failed because of, for example, incorrect timing of coitus.
- the present invention can be a valuable aid in family planning, it serves an important humani- tarian purpose.
- a ⁇ cording to the present invention the woman moistens a cotton swab with vaginal fluid and then brings it into contact with a bibulous mat containing an antibody against EIP. The mat is then washed and tested for the presence of peroxidase with a hydroperoxide and _a chromogenic substrate of peroxidase.
- the hydroperoxide may be hydrogen peroxide or a hydroperoxide generating system, such as the inorganic peroxides, sodium peroxide, barium peroxide, strontium peroxide, sodium perborate, and the like, or organic hydroperoxides, such as methyl hydroperoxide or ethyl hydroperoxide.
- Many compounds, for example, sodium peroxide, barium peroxide, strontium peroxide, sodium perborate, and the bis (1-hydroxyalkyl) peroxides generate hydrogen peroxide when moistened. Enzymatic reactions such as the action in air of L-amino oxidase on L-amino acids also generate hydrogen peroxide.
- the word hydroperoxide as used herein and in the claims is meant to include all of the compounds and types of com- pounds of this type including the hydroperoxide generat ⁇ ing compounds and enzyme systems which generate hydrogen peroxide.
- Chromogenic peroxidase substrates or chromogens which may be employed in the present invention include the following substances :
- Monoamines such as aniline and its derivatives, orthotoluidine, para-toluidine, etc.
- Diamines such as ortho-phenylenediamine
- Phenols such as phenol per se, thymol, ortho, meta and para-cresols , alpha-napthol, p,p- dihydroxybiphenyl, -phloroglucinol and guaiacol;
- Aromatic acids such as salicylic, pyrocate- chuic and gallic acids
- Leucodyes such as leucomalachite green (to produce malachite green) and leucophenol- phthalein (desirably employed in an alkaline medium) ;
- Colored dyes such as 2,6 dichlorophenol indo- phenol;
- Various biological substances such as epine- phine, the flavones, tyrosine, dihydrophenyl- alanine (producing an orange-reddish color) and tryptophane.
- Nadi reagent produc ⁇ ing a bluish color
- bilirubin producing a greenish color
- iodides which produce a brown color and, if starch is present, produce a deep blue color which is much stronger than iodide alone
- Nadi reagent is such a mixture, namely naphthol and p- phenylenediamine, which gives a better final color than the individual components.
- Another example is a mixture of 4-amino antipyrine and 1, 7-dihydroxynaphthaline.
- chromogens notably 3, 3 ' , 5 ,5 ' tetra- methyl benzidine, orthotolidine and p,p'-biphenol give a more intense color if halogen ions, such as iodide and bromide ions, or if halogenoid ions, such as thiocyanate and selenocyanate ions, are present.
- One of the preferred chromogens of the invention comprises 3, 3', 5, 5' tetramethyl benzidine and potassium thiocyanate; this mixture yields an intense blue color in a positive test.
- Another preferred chromogen is a mixture of p,p'-biphenol and potassium thiocyanate.
- Substrates which change their fluorescence in the presence of a peroxidase and hydrogen peroxide include a loss of fluorescence of scopoletin or a production of fluorescence with, for example, di ⁇ hlorofluorescin or homovanillic acid.
- Chemiluminescence is produced in the presence of peroxidases and hydrogen peroxide for the following typical substances, Luminol, zinc tetra phenylporphyrine and the like.
- Antibodies against human vaginal fluid EIP may be prepared by injecting rabbits with human vaginal fluid EIP purified according to the procedures described by E. R. DeSombre and C.R. Lytlle in Cancer Research,
- the said human vaginal fluid sample is used.
- the injections into rabbits take place at regular intervals together with Freund's complete adjuvant in a manner well known to those skilled in immunology.
- antisera are collected.
- the anti- sera may be treated by methods well known to immunolo- gist to obtain globulin samples which are enriched in EIP antibodies.
- antibody denotes pure antibody against EIP, puri ⁇ fied antibody against EIP, solutions comprising anti ⁇ body against EIP, mixtures comprising antibody against EIP, antisera against EIP and also includes antibody against the apoenzyme of EIP. Not only will antibodies against EIP function in the invention, but also anti ⁇ bodies against the apoenzyme of EIP will function in this invention and should be understood to be included herein and in the claims.
- lymphocyte hybridomas instead of injecting rabbits in the aforestated procedure for preparing the antibody to EIP, the tech ⁇ nique involving lymphocyte hybridomas may be employed. Lymphocyte hybridoma techniques are described in the book entitled Lymphocyte Hybridomas published by Springer Verlag Berlin, Heidelberg: New York, 1978. The tech ⁇ nique for making monoclonal antibodies by the technique of lymphocyte hybridomas is described in detail in the chapter by M. M. Trucco, J. W. Stocker, and R. Ceppelini in the aforesaid book, where in place of human lympho- blastoid cells employed by the aforesaid workers, puri ⁇ fied EIP from human vaginal fluid samples or the apo ⁇ enzyme of it may be used. Monoclonal antibodies against different antigenic determinants in EIP or its apoenzyme may be mixed to effect a precipitating antibody. Either monoclonal or mixed monoclonal antibodies may be employed in the present invention.
- Some animals may produce species specific EIP which may be similar enough so that the antibodies to it may cross-react with human vaginal fluid EIP.
- animal vaginal fluid EIP may be used to produce anti ⁇ bodies which may be used in place of the human antibody to EIP.
- Tissue cultures of human estrogen-sensitive tissue, such as human endometrium and the like, may pro- prise the apoenzyme of EIP, in which case EIP apoenzyme from such tissue cultures may be used as a source of apo ⁇ enzyme which may be used in the present invention.
- the apoenzyme of human EIP may be produced by the chemical synthesis of DNA and recombinant DNA methods well known to those skilled in the art. Expression in
- E. Coli of chemically synthesized genes for human EIP or its apoenzyme may be carried out as described by D. V. Goeddel et al. in Proc. Nat. Acad. Sci. USA, Vol. 76,
- a preferred embodiment of the present invention comprises antibody to EIP attached to a solid support.
- solid support may be in the form of a film, spheres, beads, tubes, ion exchange materials (such as glass beads com ⁇ prising arylamino groups or comprising carboxyl groups such as produced by Corning Glass Works) , filter paper loaded with ion exchange resins (such as produced by Whatman Paper Company) , or paper loaded with Duolite ion exchange resin (the resin being made by Diamong Shamrock Company) .
- Anti ⁇ body may be covalently linked to solid supports which comprise aryl amino groups or which comprise carboxyl groups by methods well known in the art.
- the aryl amino groups on solid supports may be diazo- tized with nitrous acid (sodium nitrite and freshly added hydrochloric acid) , washed with water and then treated with a solution comprising the antibody in sodium bi ⁇ carbonate solution.
- Antibody may be coupled to carboxyl groups on solid supports by means of the carbodiimide reaction.
- Example 1 Vaginal samples are taken daily starting on day 5 of a woman's menstrual cycle, where day 1 is taken as the day when menstruation began. Such vaginal samples can be obtained using a standard six inch cotton-tipped
- the swab while still moist, is then contacted with a paper to which was affixed an antibody to estrogen- induced peroxidase. After contact for 10 to 30 minutes, the paper was washed in water.
- the still moist paper containing the antibody is pressed against starch- iodide test paper and the test paper is moistened with 0.01% hydrogen peroxide. A strong blue color is re ⁇ lodged as a positive test and weak or no coloration is regarded as a negative test.
- day 5 or day 6 for the daily routine.
- the first positive test may be taken to indicate that ovulation is impending and will follow within two or three days.
- filter paper impregnated with 3,3' ,5,5' tetramethylbenzidine and potassium thiocyanate, impregnated with p,p' biphenol and sodium thiocyanate, or guaiac, or orthotolidine or other chromogenic substrates may be used.
- the "Hemoccult" test of Smith Kline and French Laboratories, which according to the manufacturer consists of paper impregnated with orthotolidine, may also be used.
- the vaginal fluid sample is obtained by directly contact ⁇ ing the vaginal wall with a piece of moist filter paper to which an antibody to estrogen-induced peroxidase was attached.
- the filter paper was affixed toward the end of a plastic strip about 0.012 inches thick, 0.20 inches wide and 8 inches long and made of cellulose acetate.
- the paper is removed from the vagina after 20 minutes and was then washed with water. While still moist the paper is contacted with the occult blood detector part of the commercially available "Hemicombi- stix" (Ames Company) , which according to the manufacturer comprises orthotolidine and cumene hydroperoxide. •
- a positive test i.e., strong blue coloration indicates impending ovulation.
- the occult blood peroxidase detector manufactured by Boehringer Company may be used, which detector, according to the manufacturer, contains ortho ⁇ tolidine and dimethyl dihydroperoxy hexane.
- starch-iodide paper containing dry sodium perborate may be used.
- Example 3 In place of the plastic strip in Example 2 in which the antibody to estrogen-induced peroxidase is toward one end and a separate occult blood detector is provided on another strip, a strip was made with both the anti ⁇ body and the blood detector as parts of the same strip, the antibody being at one end and the detector at the other end. The contact between the two was achieved by folding the strip at its middle and holding the ends in contact after the antibody end had been contacted with the vagina and had been washed.
- Example 4 A swab similar to that of Example 1 and containing vaginal fluid is dipped into a 1% solution of sodium chloride contained in a test tube. Affixed to the inner wall of the test tube. Affixed to the inner wall of the test tube is an antibody to estrogen- induced peroxidase. After 30 minutes the swab is
- Example 5 A swab similar to that of Example 1 and containing vaginal fluid can be dipped into a solution containing 0.1 milligram per milliliter of rabbit antisera against estrogen-induced peroxidase. After 30 minutes activated charcoal is added. It is occasionally stirred, and the suspension is filtered. To the filtrate is added 0.001% final concentration of 3,3 ',5,5' tetramethyl ⁇ benzidine and 0.1% final concentration potassium iodide, to which a 0.005% final concentration of hydrogen peroxide is freshly added. A blue color is a positive indication of estrogen-induced peroxidase in the vaginal sample.
- Example 6 A swab similar to that of Example 1 and containing vaginal fluid can be dipped into a solution containing 0.01 milligram per milliliter of mixed clonal antibody to estrogen-induced peroxidase as described herein. The mixture is centrifuged and the supernatent is allowed to stand for 12 hours. The system is centri ⁇ fuged again, the supernatent discarded, and the pellet was suspended with stirring into a 0.1% sodium chloride ' solution. After the system is again centrifuged 7 the pellet is contacted with starch-iodide paper and moistened with a 0.0005% hydrogen peroxide. A blue coloration is a positive indication of estrogen-induced peroxidase in the vaginal sample.
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Urology & Nephrology (AREA)
- General Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Endocrinology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Methode de detection de l'ovulation imminente chez la femme par des moyens de controle utilisables par tous. La methode de controle consiste a mettre en contact des echantillons de fluide vaginal avec un tampon absorbant contenant un anticorps contre la peroxydase induite par oestrogene. Le tampon est ensuite lave puis analyse pour determiner l'absence ou la presence de peroxydases.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US5081079A | 1979-06-21 | 1979-06-21 | |
US50810 | 1987-05-18 |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0030560A1 true EP0030560A1 (fr) | 1981-06-24 |
Family
ID=21967573
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP80901384A Ceased EP0030560A1 (fr) | 1979-06-21 | 1980-12-30 | Indicateur specifique d'ovulation imminente |
Country Status (2)
Country | Link |
---|---|
EP (1) | EP0030560A1 (fr) |
WO (1) | WO1980002800A1 (fr) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0126772A1 (fr) * | 1982-12-03 | 1984-12-05 | E.I. Du Pont De Nemours And Company | Analyse immunologique a support chromogene |
AU2706384A (en) * | 1983-05-06 | 1984-11-08 | Monoclonal Antibodies Inc. | Colorimetric immunoassay on solid surface |
EP0231010A3 (fr) * | 1986-01-27 | 1990-10-17 | INCSTAR Corporation | Procédé pour un essai immuno-enzymique en phase solide et essai d'hybridisation des acides nucléiques et dessin d'un bâtonnet d'échantillon et substrat chromogène stabilisé |
GB9217864D0 (en) | 1992-08-21 | 1992-10-07 | Unilever Plc | Monitoring method |
DE19957367A1 (de) * | 1999-11-29 | 2001-05-31 | Merck Patent Gmbh | Nachweis von Peroxidasen |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2295425A1 (fr) * | 1974-12-21 | 1976-07-16 | Boehringer Mannheim Gmbh | Application de derives de 3,3, 5,5-tetra-alcoylbenzidine en tant qu'indicateurs d'oxydation dans des essais rapides |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3472738A (en) * | 1967-06-12 | 1969-10-14 | Weston Lab Inc | Test implement for detecting peroxidase |
US3644177A (en) * | 1969-11-12 | 1972-02-22 | Yissum Res Dev Co | Monitoring penicillin in biological substances |
US3817837A (en) * | 1971-05-14 | 1974-06-18 | Syva Corp | Enzyme amplification assay |
US4026879A (en) * | 1976-03-23 | 1977-05-31 | Hoffmann-La Roche Inc. | Antigen-containing propranolol derivatives |
-
1980
- 1980-06-20 WO PCT/US1980/000813 patent/WO1980002800A1/fr unknown
- 1980-12-30 EP EP80901384A patent/EP0030560A1/fr not_active Ceased
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2295425A1 (fr) * | 1974-12-21 | 1976-07-16 | Boehringer Mannheim Gmbh | Application de derives de 3,3, 5,5-tetra-alcoylbenzidine en tant qu'indicateurs d'oxydation dans des essais rapides |
Non-Patent Citations (1)
Title |
---|
See also references of WO8002800A1 * |
Also Published As
Publication number | Publication date |
---|---|
WO1980002800A1 (fr) | 1980-12-24 |
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Legal Events
Date | Code | Title | Description |
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PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
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AK | Designated contracting states |
Designated state(s): AT CH DE FR GB LU NL SE |
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STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION HAS BEEN REFUSED |
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18R | Application refused |
Effective date: 19820118 |