GB1563299A

GB1563299A - Methods of pregnancy testing and a test kit therefor

Info

Publication number: GB1563299A
Application number: GB53444/76A
Authority: GB
Original assignee: Rafa Laboratories Ltd
Current assignee: Rafa Laboratories Ltd
Priority date: 1975-12-26
Filing date: 1976-12-21
Publication date: 1980-03-26
Also published as: AT352904B; JPS5282892A; BE849667A; DE2657292A1; IL48741A; IT1068738B; NL7614359A; IL48741A0; ES454542A1; ZA767607B; FR2336113A1; SE7614387L; FI763647A; AU2049576A; ATA962176A

Description

(54) METHODS OF PREGNANCY TESTING AND A TEST KIT THEREFOR (71) We, RAFA LABORAT ORIES LTD., an Israeli Company, of Ezor Hatassya Romema, Jerusalem, Israel, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- The present invention relates to an enzymatic colour test for the determination of pree;nancy.

There are known various methods for the determination of HCG. Some biological methods require the use of specific animals, for example rabbits, mice and frogs. These biological tests have serious drawbacks; they require the availability and housing of many animals meeting specific requirements, which make the method time consuming, complicated and expensive.

There are also known some immunological tests utilising certain serodiagnostic compositions (see e.g. Israel Patent Specifications Nos. 19301 and 34960).

There is also known a colour test utilizing antibody-enzyme-conjugates (see FEBS Letters, Vol. 43, No. 2, July 1974, Pages 215--217).

Both the immunological and colour tests require complicated apparatus and can be performed only in an adequately equipped laboratory and with the aid of skilled people.

However, it has become desirable to develop a test which may be performed at home so that any woman can easily determine whether she is pregnant or not. It is readily understood that such test should not require any complicated means and be performable on the basis of simple instructions.

According to the first aspect of the present invention there is provided a method for the determination of pregnancy of a woman in which method an enzyme labelled anti-HCG-IgG is admixed with a sample of body fluid of the woman and preincubated: the said mixture is then admixed with insolubilized anti-HCG-IgG in the form of a gel and, after a pre-determined period of incubation, admixed with a developing dye solution, whereafter a colour change would indicate that the woman who provided the sample is pregnant.

HCG stands for human chorionic gonadotropin and IgG for immunoglobulin G.

As enzymes for the method according to the present invention various known enzymes may be utilised. However, the preferred ones are horse-radish peroxidase, alkaline-phosphatase and glucose-oxidase.

The dye (solution) to be utilised is a function of the enzyme utilised. Thus, for example, for the horse-radish peroxidase, one preferably utilises o-dianisidine in a H202 solution. As another suitable dye, there should be mentioned benzidine.

Any suitable source may be utilized for obtaining the anti HCG-lgG as long as the material obtained is sufficiently specific.

Thus, for example, as suitable sources for obtaining said material one may consider rabbits and goats, as well as birds or other animals.

In a preferred embodiment of the invention one adds, in order to make the test more sensitive, one or both of the following substances: a. normal y-globulin obtained from any suitable source-e.g. normal rabbits, sheep or goats (to help to avoid non-specific reactions).

b. some "free" anti HCG-IgG (as opposed to insolubilised) (to help to enhance the reaction), being obtained from the same sources as the gel and the enzyme labelled anti HCG-IgG. These substances, if present, are admixed first with the enzyme labelled anti HCG-lgG.

As the body-fluid there may be utilized any fluid obtained from the woman, preferably urine.

In a preferred embodiment of the invention the entire mixture before being developed by the dye solution is washed several times with a suitable washing solution (e.g.. a borate saline solution.

The test can be perfomed in any suitable transparent vessel, e.g., a test tube. even in a bag or as a paper impregnation.

If the woman is pregnant, that is HCG is present in the body fluid, a colour being specifie to the enzyme and the corresponding dye utilised appears after the addition of the developing solution. If after about half an hour after the developing solution has been added no colour appears, this an indication that the woman is not pregnant.

According to the second aspect of the present invention there is provided a kit for carrying out the method of the first aspect, which kit comprises: (a) an enzyme labelled anti HCG-lgG: (hs meolubilized anti-HCG-IgG in the form of a gel: (c) a developing dye solution.

In a preferred embodiment, the enzyme labelled anti HCG-lgG is used with normal g-globulin and/or free anti HCGlgG.

Ingredient (a) is preferably stored and supplied in a liophilized form.

It can readily be understood that not all said ingredients have to be packed in one box. Thus, even if one or two of the above ingredients are being packed separately this is to be considered as being within the scope of the present invention.

The present invention will now be illustrated with reference to the following Examples without being limited by them.

Example I Preparation of anti HCG-lgG Normal rabbits were injected intramuscularly three limes (once a week) in the foot pad with 0.6 ml each of a suspension of HCG homogenized with an equal volume of complete Freund's adjuvant. The final concentration of HCG was 0.5 mg/ml. The potency of the HCG may vary from 28003600 u/mg and is by no means critical for the success of the iminunisation. The HCG was dissolved in a ) X5"" w/v sodium chloride solution. It may also be dissolved in any other suitable buffer solution having a pH range of 6.8-8.5.One week after the last injection the rabbits were bled and the serum separated by centrifugation, the unspecific antibodies were separated from the serum by incubation for half an hour with an equal volume of insolubilized normal human serum, and after this time the solution was centrifugated.

The specific anti llCG-lgG was precipitated from the serum by the addition of 60", (v/v) of a saturated ammonium sulphate solution (75",, w/v) to the anti HCG serum. The precipitate which was obtained was dissolved in a buffered saline solution as described and dialysed for 2-3 days against the same buffered saline solution.

The borate saline solution in this and in the following Examples was prepared as follows: I. NaCI 0.85 /" w/v (i.e. 8.5 girl) Stock Solution II. H3BO3 l2.37g NaOH 0.52 g NaCI 8.00 g H2O up to 1000 ml.

Each 97 ml of Solution I were admixed with 3 ml of Solution II.

The concentration of the anti HCG-lgG was determined by spectophotometry using w.v. at 280 my. I mglml IgG equivalent with OD 280 my 1.4.

It is important to check that the preparation obtained is free from peroxidase activity.

EXAMPLE 2 Preparation of insolubilized anti HCG-lgG as a gel The reaction was performed under the white light of a fluorescent lamp. After mixing 2.5 ml of a bisacrylamide solution, 2.5 ml of borate saline, 1 ml of proteins (containing 30-50 mg of anti HCG-IgG from Example I and another suitable protein source, e.g. normal rabbit IgG or Bovine serum albumine (BSA) having a concentration of 100 mg/ml and of 0.1-0.3 ml of riboflavin (from 0.2 mg/ml aqueous solution), a photoelectric reaction occurred.

After half an hour incubation of the above mixture at 250--360C an insoluble gel appeared. The gel was homogenized and washed 3 times in borate saline. The amount used for a test is generally from 0.1-I .0 packed gel. The bisacrylamide solution was prepared as follows: I g of bis - (N,N' - methylbisacrylamide) (GHroN202) and 4 g of acrylamide (CH2CHCONH2) were dissolved in 19 ml of distilled water.

Example 3 Preparation of enzyme labelled anti HCG IgG 5 mg of horseradish peroxidase were dissolved in 1.0 ml of 0.3 M sodium bicarbonate. 0.1 ml of fluorodinitrobenzene I)';, solution in absolute alcohol was added to said solution and the mixture was stirred gently for I hour. 1.0 ml of 0.06M sodium periodate was added and mixing was continued for 30 minutes. Then 1.0 ml of 0.16M ethyleneglycol was added and mixing continued. The mixture was then dialysed against a carbonate buffer (0.oil pH 9.5) for I day at 4"C. 5 mg of anti HCG-lgG were added and the mixture was mixed for 2-3 hours at room temperature.Then the mixture was dialysed against 0.01 (PBS) pH 7.1 at 4"C. The PBS was prepared by admixing: 17.5 ml Na2HPO4 0.15M 32.5 ml KH2PO4 0.15 M 50.0 ml saline (0.85% w/v NaCI) Approximately 90% by weight of immunoglobulin was thus labelled and 70- 90 /O by weight of peroxidase was coupled with the IgG. The labelled anti HCG-IgG was separated by passing the dialysate through a column of Sephadex G200 (Pharmacia AB). (The word "Sephadex" is a Trade Mark). The enzyme labelled anti HCG-lgG may be stored in a freeze dried form in a concentration suitable for one or more tests.The enzyme activity is measured in a spectrophotometer, visible light, wavelength 460 m,u. (0.1 ml test mixture, 2.9 ml dye incubated for 3 min. at room temperature). I drop of HCI 6N was added to stop the reaction and the result read in the spectrophotometer. The enzyme activity is expressed in mU and it is calculated as follows: A/min.

1 mU= 103 3.766 A=OD absorption EXAMPLE 4 Performance of the test The following reagents are mixed in a test tube and are pre-incubated for A hour.

a. 0.1 ml of an enzyme labelled anti HCGlgG having an enzyme activity of 65 my.

b. 0.1 ml of normal rabbit y-globulin (or sheep or goat) having a concentration of 10--100 mg/ml.

c. 0.1 ml of body fluid to be tested, preferably urine.

The addition of 0.1 mg-0.5 mg of free HCG-lgG enables the reaction to be more sensitive.

After the pre-incubation period the mixture was passed into another tube containing the packed insolubilized anti HCG-IgG gel in a volume of about 0.3 ml. This composition was left for an incubation period of 15-30 minutes. After this time, the composition was washed four times with borate saline (by centrifugation or by immersion of a nylon bag containing the mixture in the solution). After the last washing, 2.5 ml of the developing dye solution (prepared as described herein) are added, the composition is shaken well and left to stand for about 5 minutes. If HCG was present in the test body fluid, a reddish brown colour will appear; if not, a reddish brown colour will not appear.

Preparation of the dye solution 0.5 ml of a 0.3 "v/v H202 solution was put into a 0.01 M phosphate buffer having pH 6 up to a volume of 50 ml. 0.41 ml of a 1 " w/v orthodianisidine aqueous solution was then added to the above buffered solution.

WHAT WE CLAIM IS: 1. A method for the determination of pregnancy of a woman, in which method an enzyme labelled anti HCG-lgG is admixed with a sample of body fluid of the woman: and pre-incubated; the mixture is then admixed with insolubilized anti HCG-IgG in the form of a gel and, after a predetermined period of incubation, admixed with a developing dye solution, whereafter a colour change would indicate that the woman who provided the sample is pregnant.

2. A method according to Claim 1, wherein the enzyme utilized is horse-radish peroxidase.

3. A method according to Claim 1 or 2, in which the dye solution is o-dianisidine in an H202 solution.

4. A method according to any one of Claims 1 to 3, in which the enzyme labelled anti HCG-IgG is first admixed with normal y-globulin and/or free anti HCG-IgG.

5. A method according to any one of Claims 1 to 4, wherein the body fluid utilised is urine.

6. A method according to any one of Claims 1 to 5, wherein the mixture is washed before the dye solution is admixed therewith.

7. A method according to claim 6, wherein the washing solution is a borate saline solution.

8. A method for the determination of pregnancy, substantially as hereinbefore described with reference to the Examples.

9. A test kit for carrying out the method according to any of Claims I to 8, which kit comprises:- (a) an enzyme labelled anti HCG-IgG; (b) insolubilized anti HCG-IgG in the form of a gel; (c) a developing dye solution.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. against a carbonate buffer (0.oil pH 9.5) for I day at 4"C. 5 mg of anti HCG-lgG were added and the mixture was mixed for 2-3 hours at room temperature. Then the mixture was dialysed against 0.01 (PBS) pH 7.1 at 4"C. The PBS was prepared by admixing: 17.5 ml Na2HPO4 0.15M 32.5 ml KH2PO4 0.15 M 50.0 ml saline (0.85% w/v NaCI) Approximately 90% by weight of immunoglobulin was thus labelled and 70- 90 /O by weight of peroxidase was coupled with the IgG. The labelled anti HCG-IgG was separated by passing the dialysate through a column of Sephadex G200 (Pharmacia AB). (The word "Sephadex" is a Trade Mark).The enzyme labelled anti HCG-lgG may be stored in a freeze dried form in a concentration suitable for one or more tests. The enzyme activity is measured in a spectrophotometer, visible light, wavelength 460 m,u. (0.1 ml test mixture, 2.9 ml dye incubated for 3 min. at room temperature). I drop of HCI 6N was added to stop the reaction and the result read in the spectrophotometer. The enzyme activity is expressed in mU and it is calculated as follows: A/min. 1 mU= 103 3.766 A=OD absorption EXAMPLE 4 Performance of the test The following reagents are mixed in a test tube and are pre-incubated for A hour. a. 0.1 ml of an enzyme labelled anti HCGlgG having an enzyme activity of 65 my. b. 0.1 ml of normal rabbit y-globulin (or sheep or goat) having a concentration of 10--100 mg/ml. c. 0.1 ml of body fluid to be tested, preferably urine. The addition of 0.1 mg-0.5 mg of free HCG-lgG enables the reaction to be more sensitive. After the pre-incubation period the mixture was passed into another tube containing the packed insolubilized anti HCG-IgG gel in a volume of about 0.3 ml. This composition was left for an incubation period of 15-30 minutes. After this time, the composition was washed four times with borate saline (by centrifugation or by immersion of a nylon bag containing the mixture in the solution). After the last washing, 2.5 ml of the developing dye solution (prepared as described herein) are added, the composition is shaken well and left to stand for about 5 minutes. If HCG was present in the test body fluid, a reddish brown colour will appear; if not, a reddish brown colour will not appear. Preparation of the dye solution 0.5 ml of a 0.3 "v/v H202 solution was put into a 0.01 M phosphate buffer having pH 6 up to a volume of 50 ml. 0.41 ml of a 1 " w/v orthodianisidine aqueous solution was then added to the above buffered solution. WHAT WE CLAIM IS:

1. A method for the determination of pregnancy of a woman, in which method an enzyme labelled anti HCG-lgG is admixed with a sample of body fluid of the woman: and pre-incubated; the mixture is then admixed with insolubilized anti HCG-IgG in the form of a gel and, after a predetermined period of incubation, admixed with a developing dye solution, whereafter a colour change would indicate that the woman who provided the sample is pregnant.

10. A kit according to Claim 9, wherein

the enzyme labelled anti HCG-IgG is admixed with normal y-globulin and/or free anti HCG-lgG.

II. A kit according to Claim 9 or 10, wherein ingredient (a) is in a liophilized form.

12. A method of pregnancy testing, substantially as described in foregoing Example 4.

13. A kit for the performance of pregnancy testing, substantially as described in foregoing Example 4.