CA1185176A

CA1185176A - Method and kit for detecting pregnancy

Info

Publication number: CA1185176A
Application number: CA000409873A
Authority: CA
Inventors: Bernard Sulitzeanu
Original assignee: Teva Pharmaceutical Industries Ltd
Current assignee: Teva Pharmaceutical Industries Ltd
Priority date: 1981-09-16
Filing date: 1982-08-20
Publication date: 1985-04-09
Also published as: US4508829A; EP0074520B1; ES8401262A1; DE3266200D1; NZ201583A; IL63855A; EP0074520A1; BR8205357A; ATE15549T1; ZA825842B; JPS5862563A; AU8711782A; IL63855A0; ES515189A0; AU550423B2

Abstract

Abstract Method and kit for pregnancy detection adapted for self-performance. Urine to be tested is contacted with a lectin substrate being lectin bound to a solid support and capable of binding HCG. After separation of the lectin substrate from the urine the substrate is contacted with a liquid colour reagent comprising a coloured carrier material and anti-HCG antibodies bound thereto. If after separation of the coloured reagent from the substrate the latter remains coloured, the subject is pregnant, while lack of colour indicates non-pregnancy. A preferred colour reagent comprises killed and stained Staphylococci bacteria. There is also provided a kit for self-performance of the method comprising at least one column packed with a lectin substrate and adapted for the controlled passage of liquid therethrough, and a colour reagent.

Description

Method and _i for Preqnancy Detection The present invention concerns a method and a kit for early pre~nancy detection.
Pregnant women secrete soon after the irnplan-tation of a fertilized ovum in the chorionic t:issuesan increasing amount o-f human chorionic gonado-tropin (HCG) some of which is excreted in the urine. Most presently available pregnancy detection methods are~sed on the de-tection of HCG in the urine.
One group of known methods for the detection of HCG in the urine is based on radioimmunoassay.
While these methods are very reliable and sensitive they require highly sophisticated laboratory equipment and such tests cannot be carried out by the subjects them-selves.
Another group of tests known as enzyme linked immunosorbents assay (ELISA~ is based on an enzymatic reaction associated with HCG. In accordance with that method a solution of an anti-HCG antibody labelled with an enzyme is mixed with a urine sample to be tested.
The mixture obtained is then mixed with anti-HCG anti-body associated with a carrier.

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In the absence of HCG in the urine, subs~quent washings and centrifugations of the substrate will remove the anti-HCG antibody with the enzyme linked -thereto.
Accordingly, addition to the residue remainin~ in the vessel of a non-coloured solution capable of developing a colour in the presence of the enzyme will in this case not give rise to the development of any colour. IE, however, HCG i.s present the enzyme will be retained in the residue. In that case the addition of the said solution will. give rise to the development of a colour.
This and similar methods have however not been commercialised, apparently for the reason that they are no-t sufficiently sensitive and reliable.
Another group of known methods is based on agglutination that occurs upon reaction of anti-HCG
antibodies adsorbed on a carrier such as latex particles, and HCG present in -the test fluid~ or the prevention of agglutination of HCG sensitized latex particles or red cells. In the direct, agglutination method anti-HCG
antibody bearing material is contacted with the tested urine and if the latter contains HCG there occurs an agglutination which can be detected visually. In accordance with the indirect, agglu-tination inhibi-tion method anti-HCG antibodies are contacted wi-th the tested urine and subsequently with a reagent comprising llCG
cleposited on gel particles or on red blood cells. If the urine contains HCG the latter reacts with the anti-HCG
antibodies with the consequence that the subsequently added HCG reagen-t does not cause agglutination. If, however, the tested urine does not contain any HCG the anti-HCG and HCG reagent react with each other wi-th consequential agglutination.

Such methods are described, for example, in U.K patent ~o. 1,561,920 (Warner Laboratories3, Israel patent No. 50929 (American ~ome Products ~orporation) and Israel patent No. 47223 (Rafa Laboratories).
Some of these methods have become commercial and are even used for do-it-yourself testing. However, these methods have some drawbacks in that they are of limited sensitivity, not sufficiently reliable in that they produce a relatively high proportion of false positive and false negative results, and cannot be employed in the very early stages of pregnar.cy.
The early and reliable detection of pregnancy is of great importance. Thus, where for some reason the pregnancy is undesired and has to be interruptecl it is important to establish the pregnancy in as early a stage as possible. In other cases, where the woman is in the habit of taking certain drugs which may be teratogenic, it is important to know of the pregnancy as early as possible so that the taking of such drugs may be inter-rupted. In still other cases where it is known thatthe woman will suffer from certain ailments in consequen-ce of pregnancy, e.g. an inability to hold the fetus under normal conditions, adequate treatment has to be initiated as soon as possible.
As a rule women are reluctant to go to laboratories for testing at an early stage of a missecd period, be it because of the trouble that this involves or be it for psychological reasons.

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For all -these reasons it is of great importance to provide a simple and reliable method Eor the earliest possible self-determination of pregnancy. It is the object of the present invention to provide such a method and a ki-t therefor.
In accordance with -the present invention there is provided a method for the detection oE pregnancy comprising:
i) contacting urine of a tested subject wi-th a lectin bound to a solid support (lectin substra-te) and capable of binding HCG;
ii) separating the lectin substra-te from the urine;
iii) contacting the lectin substrate with a liquid reagent comprising a coloured carrier material and anti-HCG antibodies bound thereto (colour reagent); and iv) separating the colour reagent from the lec-tin substrate.
The liquid colour reagent will as a rule be in form of an aqueous suspension.
During the contact between the lec-tin substrate and the -tested urine any HCG presen-t in the urine is bound by the lectin. During the subsequent con-tact between the lectin and said colour reagent, the anti-HCG antibodies react with any HCG bound to the lectin with the consequence that the coloured carrier is also bound -to the lectin.
Accordingly, if HCG was present in the -tested urine then after the separation of -the excess colour reagen-t Erom the lectin, the lectin subs-trate remains stained. If on the other hand, no HCG was present in -the tested urine none of the colour reagent is bound to the lectin so that after separation from the colour reagent the lectin substrate remains unstained.

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Thus stained lec~in substrate at the end of the test signifies pregnancy while unstained lectin substrate at the end of the test signifies non-pregnancy.
A typical lectin that can be employed in accordance with the invention is Concanavalin A (herein-after for short Con-~) which is a lec-tin extracted from the meal of Jack bean.
~ xample of other lectins that can be employed in accordance with the invention are whea-t germ lec-tin, lentil lectin and soy bean lectin.
In the performance of the new method according to the invention HCG is adsorbed by the lectin substrate and accumulates thereon and in this way HCG from a relatively large amount of urine is concentrated in a simple and effective way. Consequently a relatively large amount of HCG is subsequently brought into reaction with the anti-HCG antibody and in this way the sensitivity of detection is increased several fold as compared -to commercially available, non-isotopic techniques.
A significan-t innovation and depar-ture that characterizes the invention is the use of a pre-s-tained colour reagent and the absence of any in-situ colour reaction. Consequently the presence or absence of HCG
in the tested urine can be read unmistakeably with the naked eye by the presence or absence of colour on -the lectin substrate.
In a preferred embodiment of the invention the colour reagent comprises killed and staine~ Staphylococci bacteria of the kind that when alive produces protein A, ~5~

coa-ted with anti-HCG antibodies. SGme Staphylococci bacteria produce protein A which is capable of binding immunoglobulins, including anti-HCG antibodies, and this protein remains present in the killed bacteria.
secause of their content of protein A and the ability of that protein to interact with immunoglobu-lins ~IgGs), Staphylococci bacteria are widely used as an immunological reagent. Thus, protein A binds with very high affinity (2 x 108 l~mole) to over 90% of the IgG of rabbits, humans and guinea pigs and to a lesser extent to the IgGs of mice, rats and other species.
Binding of protein A to IgG is extremely rapid being com-plete in a metter of seconds. These properties have been taken advantage of by the use of Staphylococci in immunoassays, for direct binding of primary immune com-plexes instead of the second antibody (see for example, Natali et al, Journal of Immunological Methods, 25 (1979), 255-264). Use is being made of these properties of Staphylococci bacteria for the purposes of the present in~ention in a manner that has never been suggested before.
Alternatively it is also possible to bind the anti-HCG antibodies with other suitable carriers such as, for example, coloured latex. However, in such cases it is necessary to perform more complicated reac-tions for binding the anti-HCG antibodies to the carrier and care must be taken that such reactions do not adversely affect the antibody.
Examples of solid supports for the lectin are various gels such as the bead-formed agarose gel Sepharose. For example, Con-A covalently bound to Sepharose*4~(8-10 mg Con-A per 1 ml packed gel), pro-duced 'oy Pharmacia Fine Chemicals, Sweden, can be used.
A similar preparation *Trademark ~5~

can be obtained by coupling Con-~ to Br-CN activated Sepharose ~ as described extensively in the literature.
In a preferred embodiment of the invention the lectin substrate is packed into a column adapted for the controlled passage oE liquids therethrough. In the performance of the method with such a column the urine is loaded onto the column and is then discharged. ~hereafter the colour reayent is passed through the co]umn and -this is followed by washing with a suitable buffer solution.
If at the end of the operation the column remains stained the test result is positive, i.e, the subject is pregnant.
If, on the other hand, the column remains unstained the test resul-t is negative, i.e, the subject is not pregnant.
The invention also provides a kit for -~he self-performance of the pregnancy detection method accordingto the invention, which kit comprises at least one column packed with a lectin substrate and adapted Eor the controlled passage of liquid therethrough, and a colour reagent (as herein defined).
Preferably the kit according to the invention also contains means such as a funnel and filter paper for the introduction of the test urine into the column.
Preferably the anti-HCG antibody colour reagent is packed in unit dosage form such that each such dosage is suitable for the performance of one single test.
The kit according -to this invention may also con-tain a buffer solution for washing the column prior to final reading.
The anti-HCG colour reagent may be supplied in liquid form, e.g. as a suspension in a buffer solution or in a lyophilized form together with a separately packed buffer 5:~7~

solution. In the latter case the required colour reacJent suspension is prepared before carrying out the test.
Preferably the kit according to ~he invention also contains instructions for the performance of -the pregnancy test according to the invention.
The pregnancy testing method according to the invention is sensitive, reliable and simple to perform and gives reliable results already at a very early stage of pregnancy, as early as 6 days after a missed period. The false positive results have been found -to be less than 1~ while there are practically no false negative resu]ts. The sensitivity of the test is high and less than 1 IU/ml of HCG in the urine can reliably be detected. The results are obtained very fast, e.g.
within 10-20 minutes from start to finish as compared to at least 2 hours in the known hemagglutination test.
Also the test according to the invention is not affected by vibration and other external disturbances which compares favourably with the hemaglutination tests that are affected even by unnoticed vibrations.
~ The results obtained in accordance with the nvention are illustrated in the accompanying drawing which contains graphical representations of the test results obtained from the urine of 690 out of 963 tested subjects (for the balance of 303 subjects the exact day after the missed period could not be de-termined). The grapl1s represent the number of tests as a function of days after a missed period. The drawn out line represen-ts positive test results, the d~tted line negative test results and the dash-dotted line false positive results.
There are no ~alse negative results.

5~6 A typical kit according to kh~ in~entisn compris~s the following components:
A transparent stoppered plastic column which contains 1.5 ml of Con-A Sepharose 4B
admixed with pure Sepharose 4B, the mixture having been washed with a solution of ovalbumin;
Filtéx paper;
An ampoulle containing 2 ml of acetate buffer;
An ampoulle containing 500 ~l of a colour reagent as in Example 5 hereinafter~
The procedure of performing a test with this kit is as follows:
The stoppers at the upper and lower ends of the column are opened to let the fluid that is in the column drip out until just before drying. Three ml of first morning urine is loaded onto the column through -the opening containing a filter paper and allowed -to drip out until just before drying. Then the colour reagent is loaded onto the column. This is followed by washing with

2 ml of acetate buffer and then the results are read:
If the column is stained the test is posi-tive, i.e. the subject is pregnant. ~f the column remains white the test is negative, i.e. the subject is not pregnant.
The preparation of the various components of the kit according to the invention will now be described by way of example only:
Example 1 - Preparation of the column A batch of gel for packing the column is prepared by mixing 1 volume of a 50% suspension of Con~A Sepharose 4B, 1.5 volumes of a 50~ suspension of Sepharose 4B and ~5~

1.25 volumes of acetate buffer p~I 6rO~ and the mixture is degased in vacuo. 3 ml of ~he degased mixture are loaded onto a column whose lower, discharge end is stoppered, The column dimensions are 8 x 1 cm. The column is placed in vertical position and the mixture therein is allowed to set for 1 hour at room temperature.
Thereafter the column is washed with 10 ml of acetate buffer pH 6.0 containing 1.0~ of sodium azide and 0.05%
of a surfactant known commercially as "Tween 20" (trade mark). There follows another washing with 3 ml of an aqueous solution of 2 mg/ml of ovalbumin. The column is then stoppered below and on top with some liquid remaining inside so as to cover the upper surface of the gel.
Fxample 2 - Pre~ara-tion of killed Staphylococci Staphylococcus aureaus strain 12598 are grown according to the method described by Xessler et al (J. Immunol. 117. 1482 (1976)) on Pennasay broth containing g-glycerophosphate. The bacteria are ]cilled by 1.5 hours fixation in 1.5% formaldehyde followed by heating to 80C
for 5 minutes. The killed bacteria are tested for sterility and only those batches with undetec-ted growth are used.
Example 3 - S-taining of the killed Staphylococci bacteria The killed bacteria are stained with Hematoxylin.
2 ml of wet packed bacteria are washed in borate saline solution and resuspended in 94 ml of distilled water.
One ml of 1~ solution of FeSO4.7H2O and 5 ml of 0.5%
hematoxylin are added while stirring. The stirring is continued overnight in the cold~ The bacteria are collected by centrifugation, washed several times with bora-te saline and once with a borate buffer saline solution containincJ
0.1~ by weight of bovine serum albumin, 4~ by weight of sucrose and 0.1~ by weight of sodium azide. This is followed by homogenlzation in a pes-tle and a tube homogenizer in the same borate saline solution used above so as to obtain a 10% by weigh-t of a blue coloured suspension.
Example 4 - Preparation of anti-llCG antibodies a) Immunization procedure Rabbits of either sex are immunized according to -the method described by Voitukaitis et al, J. Clin.
Endocr. 33, 988 (1971) with either purified HCG (11,000 IU/mg) or by partially purified HCG (3,000 IU.mg)O The rabbits are boosted every month until a hemagglutinating titer of at least 1:~,000 is obtained, which- usually takes at least three months from the first injection. The rabbits are then bled, the serum separated and subjected to a purification process as described hereinafter.
b) Removal of interfering_antibodies (i) Preparation of polymerized normal human serum (NHS) _ _ 10 ml of NHS is admixed with 1 ml acetate buffer pH 5.0 and 3 ml of a 2.5% glu-taraldehyde solution in a 0.15 M phosphate buffer solution. The mixture is stirred for 20 minutes and then left to stand ~or 3 hours at room temperature. The gel forming is homogenized and washed 3 times by centrifugation.
(ii) Absorption of anti-HCG antibody serum on NHS __ __ One volume of the above pellets is admixed with one volume of the HCG antibody serum obtained in part a) of this example and the mixture is modera-tely stirred for 1 hour at room temperature. 'rhis is followed by centrifugation. The partially purified supernatant serum is separated and the precipitate is discarded.

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~) Removal of Con-A binding immunoglobulins 10 volumes of the serum obtained in part b) of this Example are mixed with one volume of Con-A Sepharose obtained from Pharmacia Fine Chemicals, Sweden, and the mixture is gen-tly stirred for one hour. This is followed by filtering through a sintered glass filter and the eluant is collected while the precipitate is discarded.
d) Preparation of specifically purified anti-HCG antibodies _ (i) Preparation of HCG immunosorbent Ultragel AcA34 ILKB) is used as -the carrier.
The gel is washed with water, incu~ated overnigh-t a-t 37C
in a 6% by volume glutaraldehyde solution in 0.1 M phosphate buffer pH 7.4 and is then washed extensively with bi-distilled water.
The washed gel is incubated overnigh-t at room temperature (or for 48 hours at 4C) wi-th an equal volume of a solution of 4 mg/ml of HCG in 0.1 ~ phosphate buffer, pH 7.4 At the end of the incubation the gel is washed several times with phosphate buffer saline. Columns of 5 ml immunosorbent are prepared for fur-ther use.
The gel is then washed with an eluting medium made of equal volumes of glycine-HCl buffer 0O2 M
(vol/vol) pH 2.8 then again with phospha-te buffer saline (PBS) and then with PBS containing 0.1~ sodium azide.
The gel obtained in this way is kept in the cold and it is good for several purification cycles.
(ii) Adsorption on HCG immunosorbent The anti-HCG antibody serum obtained under (i) above is passed twice in a very slow flow through a HCG immunosorbent column obtained in accordance with part i). The column is washed repeatedly with phosphate 5~76 buffer saline till an optical density of 0,05 or less at 280 ~m is obtained in the eluant. The column is cooled in a refrigerator and washed with cold bi-distilled water. The elu-tion of -the adsorbed anti-bodies is achieved by passing through the column in thecold 7 ml of glycine-HCl buffer of pH 2.8 con-taining 0~05~ of bovine serum albumin, at a slow rate of flow.
The eluant is neu-tralized immediately with an aqueous solution of tris(hydroxymethyl)aminomethane 0.2 M and NaCl 0.5 M, pH 8.5 (Tris buffer), in order to prevent the an-tibodies from becoming denaturated by prolonged exposure to acidic condition.
This is followed by a second elution with 10-l4 ml of 0.1 M HCl containing 0.05% of bovine serum albumin and 0.5 M NaCl which is again passed through the column at a slow rate of flow.
The fractions so obtained from -the column are each neutralized with Tris buffer, then washed with phosphate buffer saline and kept in a phosphate buffer saline containing 0.1% by weight of sodium azide for further use.
The recovery of purified an-ti-HCG an-ti-bodies is from 25 to 40%.
e) Removal of undesired antibodies by adsorption or urinary proteins immunosorbent (i) Preparation of insoluble urinary protein The commercial HCG employed in par-t a) of -this Example is derived from the urine of pregnant women.
This urine contains also other proteinaceous material and consequently the rabbit develops also antibodies for such other material in addition to the desired HCG antibodies.

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Consequently a further purification is required to remove these undesired antibodies and to obtain pure anti-HCG antibodies.
For this further purification male urinary protein is used as adsorbent. This urinary protein is precipitated from whole urine by the addition of 776 g of ammonium sulfate per liter of urine while stirring for 1 hour at ~C, followed by centrifugation at 15,000 rpm for 10 minu-tes. The precipitated protein is dissolved in a small quantity of phosphate buffer saline amountlng to approximately 2.5~ of the initlal volume of urine.
The dissolved proteins are then put in a dialyzing bag and dialysis is carried ou-t in l5 mM of phosphate buffer saline for several days in the cold with 3 daily replacements of the buffer solu-tion. During dialysis some of the urinary proteins precipita-te ou-t.
To 0.5 ml of the insoluble urinary protein so obtained 8 ml of phosphate buffer saline containing 0.1~ by volume of glutaraldehyde are added.
The mixture is stirred for 1 hour at room temperature and is then centrifuged. The resultant pellets are washed twice with phosphate buffer saline and are then mixed with 7 ml of an 0.2 M aqueous glycin solution pH ~.5 and the mixture is left to stand overnigh-t for blockin~
excess glutaraldehyde. The mixture is then thoroughly washed with phosphate buffer saline until the proteins are no longer detected in the washing solu-tion. The pellets are then suspended in phosphate buffer saline a-t a 5% concentration.
tii) Absorption on insoluble urinary proteins 2 ml of the purified anti-HCG antibodies obtained according to part d) of this ~xample are adsorbed on 1 ml of the above urinary protein immuno-sorben~ pellet suspension and the mixture is lef-t to stand 7~

for 1 hour a.t room temperature and is then filtered on a sintered glass filter. The filtrate contains purified antibodies suitable for further use in accordance with this invention.
Example ~ap~y_ cooccanti-HcG antibodies to One volume oE Staphylococci bacteria killed and stained as described in Examples 2 and 3 are mixed with one volume of an appropriate titer adsorbed anti-HCG anti.bodies obtained in accordance with Example 4 and the mixture is left to stand :Eor 1 minute at room temperature. The mixture is then dilu-ted wlth 10-12 volumes o~ a borate buffer saline solution containing 0.1% bovine serum albumin, 4% hy weigh-t of sucrose and 0.1% by weight of sodium azide and the resulting mixture is the desired colour reagent according to the invention.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed, are defined as follows:

1. A method for the detection of pregnancy comprising:
i) contacting urine of a tested subject with a lectin substrate comprising a lectin bound to a solid support and capable of binding HCG, ii) separating the lectin substrate from the urine, iii) contacting the lectin substrate with a liquid colour reagent comprising a coloured carrier material and anti-HCG antibodies bound thereto, and iv) separating the colour reagent from the lectin substrate.

2. A method according to claim 1, wherein the lectin is Concanavalin A.

3. A method according to claim 1, wherein the lectin is selected from the group consisting of wheat germ lectin, lentil lectin and soy bean lectin.

4. A method according to claim 1, wherein said solid support for the lectin is a gel.

5. A method according to claim 4, wherein said gel is SEPHAROSE.

6. A method according to claim 1 wherein said colour reagent comprises killed and stained Staphylococ-ci bacteria.

7. A method according to claim 1, wherein said colour reagent is in form of an aqueous suspension.

8. A method according to claim 1, wherein said lectin substrate is packed into a column adapted for the controlled passage of liquid therethrough.

9. A kit for the self-performance of the pregnancy detection method as defined in claim 1 com-prising at least one column packed with a lectin substrate and adapted for the controlled passage of liquid therethrough, and a liquid colour reagent com-prising a coloured carrier material and anti-HCG anti-bodies bound thereto.

10. A kit according to claim 9, wherein the colour reagent is packed in unit dosage form.

11. A kit according to claim 9, also comprising at least one vessel with buffer solution.

12. A kit according to claim 9, wherein the said colour reagent is in form of an aqueous suspension contained in at least one suitable vessel.

13. A kit according to claim 9, wherein the colour reagent is present in lyophilized form together with a separately packed suitable buffer solution for the preparation of a colour reagent, in form of an aqueous suspension.

14. A kit according to claim 9, containing instructions for the performance of the pregnancy test.