IL33186A - Method and agent for determining pregnancy - Google Patents

Description

METHOD AND AO-SWT FOR DETERMINING PREGNANCY JULIUS SCHMID, INC CASE DP 27li22 This invention relates to a diagnostic dye indicator for n 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 testf complicated test procedures requiring careful following Of instructions, complicated procedures required for preparing the diagnostic reagents, lack of sensitivity and specificity of the tests and lack of r stability of the reagents. Moreover, some of the in vityb 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. ¾■ provide an rn 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.

In accordance with the present invention, it has been discovered that certain dyes having a pH between about .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'»5" dibromo—-cresolsulfon^phthalein ) solution containing a buffering agent. However, a closely related dye, namely Chlorophenol Red (3'»3" dichlorophenolsul-fonephthalein ) can also be used. When the 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 pregnanto If the pH of the dye is below about .5 and above about 7 the diagnostic reagent does not provide satisfactory results.

In accordance with tho prooont invontlon tihoro io . ne can prepare .the aloo providod a method of proparing jy diagnostic dye reagent by hiofo-oenippiooo dissolving Bromocresol purple or Chlorophenol red in an aqueous alkali solution. ®ne includes A preferred proooduro or proparing tho dyo roagont maintain ■lo to-inoludo a buffering agent in the solution to-inopoaoa above range the/pH otability of the solution during extended periods of storage. The selection of the buffering agent is not critical. Suitable materials include phosphates, glycine, borates, etc.

Example 1 invention may be prepared as follows: To 0.1 0 grams of Bromocresol purple in a glass mortar, there was added a . oiN sodium hydroxide solution in increments of about 10 ml. while vigorously stirring, until a total of 7I4. ml. has been added. Stirring is continued until solution is completed. Thereafter about 725 ml. of a phosphate buffer (pH 6) solution ( 0 ml. 0.2M potassium phosphate monobasic solution mixed with 28 ml. 0.2M sodium hydroxide solution and diluted to 1000 ml. with water) was thoroughlymixed 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 grams of Bromocresol purple in a mortar with 18·5 cc 'a of 0.01N sodium hydroxide to produce a homogenous mixture. Thereafter this mixture is diluted to 250 cc's. 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 sufficiently 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 cc's. 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. Example 2 The diagnostic dye reagent may also be impregnated into a suitable substrate which may be composed of a cellulose containing material or other absorbent material. A suitable impregnation procedure comprises dipping strips of Whatman #1+ filter paper (each 1 cm wide and 9 cm long) into a dye solution prepared in the manner previously described for a short period of time (e.g. 1 to 2 seconds) after which the impregnated strips are dried in an oven at 50° C for one hour. After removal from the oven the strips which are yellow-green in color may be cut into any desired size.

The substrate treated with the dye may then be used by contacting it with female urine and observing the color change on the substrate. A blue-violet to deep purple indicates a positive test while a yellow-green, green or- blue-green color indicates a negative test.

The efficacy of the diagnostic dye reagent of the invention has been demonstrated experimentally on a substantial number of patients under medical supervision. To date these tests have shown a reliability of between 90 and 95$· 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.

Claims (3)

1. 33186/2 CASE DD-27l22 What we claim is : 1. A diagnostic dye reagent for pregnancy . detection comprising a Bromocresol purple or chlorophenol red solution having a pH between 5·5 and 7. and containing a buffering, agent.

2. A reagent according to claim 1, wherein the. buffering agent is a phosphate buffer. ... · . ' according to Claim 1

3. A diagnostic indicator/which comprises a Bromocresol purple .or Chlorophenol red solution haying a pH between 5·5 and 7 impregnated on a substrate. J+. A reagent or diagnostic indicator according to any one of claims 1 to 3i wherein 5. A method of preparing a which comprises dissolving Bromocresol purple or Chlorophenol red in an aqueous alkali solution and adding a buffering agent to the solution so that the resulting solution has a pH between 5·5 and 7. 6. A method according to claim 5» wherein the alkali is an alkali metal hydroxide. 7. A method according to claim 5 or 6, in which the resulting pH. is about 6. 8. A method of producing a diagnostic dye reagent aocording to claim 5» substantially as herein described with reference to Example 1. 9. A diagnostic dye reagent when prepared by the process according to any one. of claims 5 to 8. 10. A diagnostic indicator according to claim 9» in which the dye reagent is impregnated on a substrate. 33186/2.« substantially as herein described with reference to Example 1 or 2. 12. A diagnostic indicator according to claim 1, substantially as herein described with reference to Example 3· 13. , A method for pregnancy detection which comprises adding to the urine of the female subject a dye reagent according to any one of. claims 1 to 1+ and 9 to 12. ll. A method for pregnancy detection according to claim 13, substantially as herein described with reference to Example or