US 3595620 A
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United States Patent 3,595,620 PREGNANCY DETECTION Harry W. Gordon, Bronx, N.Y., assignor to Julius Schmid, Inc., New York, N.Y. N0 Drawing. Filed Oct. 28, 1968, Ser. No. 771,363 Int. Cl. G01n 21/06, 31/22, 33/16 US. Cl. 23230 8 Claims ABSTRACT OF THE DISCLOSURE A method of in vitro pregnancy detection comprises contacting a urine sample of a female with a diagnostic composition consisting essentially of Bromocresol purple or Chlorophenol Red and observing the color produced.
This invention relates to a diagnostic dye indicator for in vitro pregnancy detection.
It is known that human chorionic gonadotrophin (hereinafter referred to as HCG) is produced during pregnancy by the placenta and is found shortly after nidation in serum and urine. HCG is secreted in the urine of pregnant women and is not found in the urine of non-pregnant women except non-pregnant women afflicted with certain pathological conditions. A substantial number of the prior art pregnancy tests are based on in vivo and in vitro methods of determining the presence of HCG. Most of these tests have numerous disadvantages or drawbacks among which are: inordinate length of time required for the test, complicated test procedures requiring careful following of instructions, complicated procedures required for preparing the diagnostic reagent, lack of sensitivity and specificity of the tests and lack of stability of the reagents. Moreover, some of the in vitro tests known in the art require the maintenance of a large supply of animals of the required age and the need for multiple injections.
Accordingly, there has now been discovered a quick and simple procedure for in vitro pregnancy detection based on the use of an indicator dye which overcomes numerous disadvantages associated with the prior art pregnancy test procedures.
Thus, one aspect of the present invention is to provide an in vitro method of determining pregnancy in women and other female mammals which does not require any laboratory equipment or any special skills for interpretation of the results and which can be routinely carried out by a layman.
Another aspect of the present invention is to provide in vitro diagnostic reagents which are characterized by a high degree of sensitivity and provide accurate results in substantially faster time than heretofore possible.
An additional aspect of the present invention is to provide a diagnostic dye reagent for determining pregnancy which consists essentially of a buffered Bromocresol purple solution having a pH between about 5.5 and 7.
A further aspect of the present invention relates to a process of producing diagnostic dye reagents of this invention.
These and other aspects of the present invention will become apparent from the following description.
In accordance with my invention, it has been discovered that certain dyes having a pH between about 5.5 and 7 and preferably between about 6 and 6.5 will act as an indicator when added to female urine thereby providing a simple and accurate test for pregnancy detection. The preferred dye used as a diagnostic reagent is Bromocresol purple (',5" dibromo-o-cresolsulfonephthalein) solution containing a buffering agent. However, a closely related dye, namely Chlorophenol Red (3',3" dichlorophenolsulfonephthalein) can also be used. When the 3,595,620 Patented July 27, 1971 ice Bromocresol purple solution is added to a sample of female urine it has been found to turn a deep purple in the urine of a female who is pregnant and remain red to reddish-brown in a urine sample of a patient not pregnant. If the pH of the dye is below about 5.5 and above about 7 the diagnostic reagent does not provide satisfactory results.
A preferred procedure for preparing the dye reagent is to include a buffering agent in the solution to increase the pH stability of the solution during extended periods of storage. The selection of the buffering agent is not critical. Suitable materials include phosphates, glycine, borates, etc.
A Bromocresol buffered solution used in my invention may be prepared as follows: To 0.40 gram of Bromocresol purple in a glass mortar, there was added a .01 N sodium hydroxide solution in increments of about 10 ml. while vigorously stirring, until a total of 74 ml. has been added. Stirring is continued until solution is completed. Thereafter about 725 ml. of a phosphate buffer (pH 6) solution (250 ml. 0.2 M potassium phosphate monobasic solution mixed with 28 ml. 0.2 M sodium hydroxide solution and diluted to 1000 ml. with water) was thoroughly mixed with the Bromocresol dissolved in the sodium hydroxide to provide a buffered solution having a pH of about 6.
The in vitro diagnostic dye reagent may also be prepared by grinding 0.1 gram of Bromocresol purple in a mortar with 18.5 ccs. of 0.01 N sodium hydroxide to produce a homogenous mixture. Thereafter this mixture is diluted to 250 ccs. with distilled water to provide a red solution having a pH of about 6. It has been found that if the dye is added directly to the water and the sodium hydroxide separately added, the resulting dye solution will not produce a sufiiciently distinct color change when added to urine of a pregnant woman.
The procedure for adding the diagnostic dye reagent to the urine sample is as follows: One to two drops of the diagnostic dye reagent are added to 3 to 5 ccs. of urine in a test tube which is shaken. The color development which takes place almost instantaneously is then observed. -As previously indicated a purple color indicates a positive reaction and a red to reddish-brown color a negative reaction. The quantity of diagnostic dye reagent used is not critical. Any substantial variation in the quantity of dye added to a given quantity of urine affects only the intensity of the color observed.
The eflicacy of the diagnostic dye reagent of my invention has been demonstrated experimentally on a substantial number of patients under medical supervision. To date these tests have shown a reliability of between and False positive readings have been observed in a few instances where the female is taking a certain type of steroid containing drug. The purple color observed with these false readings is substantially lighter or fainter than the intense purple color produced in those cases where a positive reaction is observed in female urine containing HCG.
Hence, the diagnostic dye reagent may be useful as a tool for the in vitro detection of steroids and therefore permit a female who is taking an oral contraceptive to readily determine whether she has missed taking a pill in the prescribed cycle.
While the invention has been described with reference to the specific embodiments, it is to be understood that it is not to be limited thereto, but is to be construed broadly and restricted solely by the scope of the appended claims.
What is claimed is:
1. A method of in vitro pregnancy detection in a female which comprises adding to urine derived from said female a sufiicient quantity of a composition consisting essential- 1y of a Chlorophenol Red solution having a pH between about 6 and about 6.5 and observing the color produced in said urine.
2. A method of in vitro pregnancy detection in a female which comprises adding to urine derived from said female a sufficient quantity of a composition consisting essentially of a Bromocresol purple solution having a pH between about 6 and about 6.5 and observing the color produced in said urine.
3. A method of detecting pregnancy in a female which comprises contacting urine derived from said female with a diagnostic composition selected from the class consisting of Bromocresol purple and Chlorophenol Red and observing the color produced, said diagnostic composition having a pH between about 5.5 and about 6.5.
4. A method according to claim 3 wherein said diagnostic composition contains a buffering agent.
5. A method according to claim 3 wherein said diagnostic composition is Bromocresol purple.
6. A method according to claim 5 wherein the pH of said Bromocresol purple diagnostic composition is about 6.
7. A method according to claim 6 wherein said diagnostic composition is in the form of an aqueous solution.
8. A method of in vitro pregnancy detection in a female which comprises adding to urine derived from said female a sufiicient quantity of a composition consisting essentially of Bromocresol purple and a phosphate buffer, said composition having a pH of about 6, and observing the color produced in said urine.
References Cited UNITED STATES PATENTS 8/1962 Collins 23230B1O 1/1967 Fossel 23230Bl0 OTHER REFERENCES MORRIS O. WOLK, Primary Examiner E. A. KATZ, Assistant Examiner US. Cl. X.R. 252408; 424-7