IL43964A - Processes reagents and means for early diagnosis of pregnancy - Google Patents

Description

DTf>ia ϊΐπακ D'ysoNi ,o»a'is .DO' nnV ο·>οη·>η&π a^ i pzy IMPROVEMENTS RELATING TO PROCESSES,' REAGENTS AND MEANS FOR EARLY DIAGNOSIS OF PREGNANCY IMPROVEMENTS RELATING TO THE PROCESSES. REAGENTS AND MEANS ,FOR EARLY DIAGNOSIS OF PREGNANCY M ABSTRACT OF THE DISCLOSURE A reagent for the early diagnosis of pregnancy comprising a substrate of suitable erythrocytes or polymer having adsorbed thereon a predetermined ratio of chorionic gonadotropin antigen and antisera therefor. The ratio is selected so as to be. insensitive to the presence of less than 1,500 I.U. per liter of chorionic gonadotropin in the urine being tested. The reagent is used by combining with a pre-deteimined quantity of the urine to be tested and after 1/2 -2 hours inspecting for agglutination, the presence of which indicates a negative result. An apparatus is also disclosed for carrying out the process.

SPECIFICATION Field of the Invention ^ The present invention relates to a new and novel" process for an early diagnosis of pregnancy and further to a modi fied reagent and improved means for implementing the process.

Background of the Invention As is known, the gonadotropic hormones or gonadotropins are hormones stimulating the gonads. In mammals, the gonadotropins are generated by the hypophysis, the placenta, and, as regards pregnant mares, also by the endometrium. The gonadotropi hormones comprise the follicle-stimulating ones (FSH), the lutein ing ones (LH) , luteotropine (LTH) and chorionic gonadotropin (GCH). It is known that the biological effects of GCH and LH are similar. It is also known that the urine of pregnant mammals contains chorionic gonadotropin of placental origin and that in women, in the midst of the menstrual cycle, the urine contains LH of hypophyseal origin of which the action causes ovulation and formation of yellow body. It is further known that the urine of the female in the pre-menopause period, that is, in the case of incipient menopause, contains gonadotropins of hypophyseal origin.

The presence of chorionic gonadotropin of placental origin in the urine constitutes one of the early diagnostic means for pregnancy. This is the reason reagents allowing early diagnosis of pregnancy were developed.

These reagents in essence are substrates on which are adsorbed antigens consisting of chorionic gonadotropins (GCH) and chorionic gonadotropin antisera; the reaction between the GCH antigen and the antiserum on the substrate results in^ a clear, yellow precipitate homogeneous in nature, i.e. the agglutinate. When there is excess of free GCH in the reactive medium — for instance when the GCH is obtained from chorionic gonadotropin from the urine of the pregnant female — there will be no reaction as above, it being inhibited by the presence of GCH in the urine, and no agglutinate will be formed.

When the substrate on which the GCH is adsorbed consists of suitably treated erythrocytes and when there is no erythrocyte-antiserum reaction, and no agglutinate is formed, the erythrocytes will settle at the bottom of the test tube as a dark-brown ring. This reaction, which is known as the WIDE & GEMZELL reaction, was described in ACTA ENDOCRINOL, 35., 261 (1960); it was applied in French Patents 1,299,171 and 1,343,189 under the name of ORGANON N.V.

When the substrate used consists of a synthetic polymer as described in French Patents 1,322,869 and 1,411,279 under the name of ORTHO PHARMACEUTICAL CORP., wherein the antigen/ antiserum reaction is inhibited by the presence of GCH in the urine being tested, there will be no agglutination reaction.

However, the reagents described in the patents above are so sensitive that the antigen/antiserum reaction will be inhibited not only when the urine being tested contains chorionic gonadotropin from a pregnant subject, that is, of placental origin, but also when such urine contains gonadotropins of hypophyseal origin as in the cases of incipient menopause and in the middle of the menstrual cycle.

Summary of the Invention It has now been found that the gonadotropins of;^ hypophyseal origin will be eliminated in the urine both for incipient menopause and for the middle of a menstrual cycle in proportions always less than 500 I.U. per liter of urine.

On the other hand, if a quantitative and early test for pregnancy is undertaken the 9th day of delay in menstruation, then, as shown by experiment, the elimination rate of the chorionic gonadotropins (GCH) in the urine will always exceed 1,500 I.U. per liter of urine if there is pregnancy.

The intermediate zone between the GCH elimination rates of 500 and 1,500 I.U. per liter of urine lends itself to no useful interpretation since the rates falling within this zone are larger than those of incipient menopause and of the middles of the menstrual cycles, while less than those allowing diagnosis of a normal pregnancy. The present invention requires that the reagent used for indicating early pregnancy be of sufficiently high threshold to eliminate any chance of error or confusion, so that absence of agglutination occurs and is observed only when chorionic gonadotropin is present in the urine of a pregnant female.

In other words, the sensitivity threshold of the present invention must exceed 1,500 I.U. of GCH per liter of urine being examined.

The necessity for a reaction threshold exceeding 1,500 I.U. of GCH eliminated per liter of urine will become clear when examining attached Fig. 1 showing a graph of GCH elimination during pregnancy, showing as abscissa the number of months between fertilization (0) and delivery (9) , and as ordinate the va^ of the GCH eliminated in the urine and expressed in I.U. of GCH plotted on a logarithmic scale.

In this graph, zone 1 shows the region of elimination of the gonadotropin of hypophyseal origin eliminated in the urine for the case of incipient menopause and for the middle of the menstrual cycle, essentially covering the range of 0 to 500 I.U. of GCH/liter of urine. Region 2 corresponds to the intermediate range essentially comprising 500 to 1,500 I.U. of GCH/lit of urine elimination. An uneven reaction takes place and a more or less pronounced ring is likely to occur when the substrate being used consists of erythrocytes. Accordingly, the test undertaken in such instances is not amenable to results allowing clear interpretation. Zone 3 corresponds to the GCH elimination rates expressed as I.U. per liter of urine during pregnancy, during which there is no agglutination, so that the reactive medium will be appreciably cleas When erythrocytes are used as the substrate they will deposit at the bottom of such medium.

Shaded zone 4 corresponds to a sketch of the elimination of the GCH during pregnancy from fertilization to birth. The straight line 5 which is parallel to the ordinate shows the optimum date for the earliest performance possible of the diagnostic test, that is, the ninth day following delay in menstruation. Point 6 formed by the intersection of the straight line 5 with the lower boundary of zone 4, and point 7 formed by the intersection of straight line 5 with the upper boundary of zone 4, respectively show the minimum and maximum elimination rates of GCH per liter of urine on the 9th day of delay in menstruation.

The object of the present invention therefore is to create a process, a reagent and means allowing early diagnosis of pregnancy, which will better satisfy practical requirements than known processes and means with the same purpose, said object being achieved in particular by the process, reagents and means of the invention, allowing a practical and reliable early diagnosis from which are eliminated any possible influences due to incipient menopause and middles of perturbed menstrual cycles. Furthermore, the present invention allows execution of the proces and use of the reagents and the means by personnel not trained in hormonology and immunology. Reading of the reactions is practical, specific and reliable, the urine being tested in such manner that virtually any ambiguity will be eliminated in interpreting the presence of pregnancy in the person under consideration.

The object of the present invention is a process for early pregnancy diagnosis in mammals in which pregnancy causes the elimination of chorionic gonadotropin of placental origin in the urine, particularly as regards the human female, making use of a reagent essentially comprising a substrate which may consist of suitably treated erythrocytes or of a synthetic polymer on which is adsorbed chorionic gonadotropin endowed with antigen properties and a substance acting as antiserum with respect to this hormone. The process is characterized in that the possibly diluted urine is made to react with a reagent such as described above and so modified as to be wholly insensitive to chorionic gonadotropin when the latter is eliminated in the urine until the rate exceeds 1,500 I.U. of GCH per liter of urine. The reactive mixture is allowed to stand from half an hour to two hours, after which time examination will take j^ace regarding presence of agglutination, or regarding whether the latter was inhibited by the presence of any chorionic gonado-tropin due to pregnancy. Proof of such inhibition is found by the deposition of a ring of erythrocytes if latter were used as substrates for the antigenic GCH.

Another object of the present invention is a reagent for early diagnosis of pregnancy, essentially comprising a substrate constituted, for instance, of erythrocytes or of synthetic polymers on which is adsorbed chorionic gonadotropin associated with a substance acting as an antiserum with respect to the latter hormone. This reagent is characterized by being so modified as to provide a selective sensitivity threshold with respect to the chorionic gonadotropin eliminated in the urine which will be higher than 1,500 Ι.υΌ of GCH per liter of urine.

In one advantageous embodiment for the reagent of this invention, the ratio of the antibody concentration in the anti-serum/antigen of the reagent is computed in such manner that the agglutination reaction will be entirely inhibited beyond a rate of 1,500 I.U. of GCH per liter of urine.

In another preferred embodiment of the reagent, the antibody concentration of the antiserum is varied by increasing the volume of the antiserum.

In another preferred embodiment of this reagent, the antisera will be selected from the rabbits' supplies of which the antibody concentration is highest„ In another advantageous embodiment of the invention relating to the reagent, the denominator of the antibody c ncentration ratio of antiserum/antigen will be varied, i.e. the amount of antigen will be changed, by varying the amount of substrate adsorbing the antigen.

In another embodiment of the invention, the amount of antigen adsorbed on the substrate may be varied.

Another object of the present invention is apparatus for the implementation of early diagnosis of pregnancy comprising: a tube or similar device containing the reagent consisting in solid form of a substrate on which is adsorbed the antigen and a GCH antiserum, a measuring means for a given volume of urine to be tested, a tube or similar device containing a diluting liquid, all appropriately supported, and a mirror allowing inspec tion of bottoms of the tubes. The apparatus further preferably includes a screen favoring viewing the reaction in the tube or similar device where said reaction takes place, by concentrating the light it generates towards said mirror.

In conformity with an advantageous embodiment of the apparatus of the present invention, the tube or similar device containing the diluting liquid is so designed that it will simultaneously constitute a measuring means for a given volume of urine to be tested.

In conformity with another advantageous embodiment of the apparatus of the present invention, the tube or similar devic holding the reagent contains a reagent which is so modified that when the urine being examined holds less than 1,500 I„U. of GCH per liter, it will cause an erythrocyte/antibody agglu*Lna-tion in the form of a clear precipitate of homogeneous nature, and when the urine examined contains more than 1,500 I.U. of GCH per liter, the agglutination reaction will not occur, and when the antigen substrate consists of erythrocytes, these will deposit as a ring, having failed to react with the antibodies in view of the inhibiting action of the GCH in the urine being examined .

A preferred embodiment of the apparatus of the inven-tion consists in associating reference images of the likely two kinds of reactions with the mirror reflecting the image of the bottom of the tube where the diagnosing reaction takes place. The reference images are used as comparison standards with the actual one obtained by the mirror and reflected from it.

Another embodiment of the apparatus of the present invention consists in associating image-fixing means for the image obtained from the reaction tube, for instance a sensitive plate or similar device, with said apparatus.

In addition to provisions described above, the invention also includes other arrangements associated with the following description.

The present invention especially applies to the processes, reagents and implementation means of early pregnancy diagnosis that are in conformity with the arrangements described above, and furthermore relates to any agent, arrangement, apparatus, design etc, that are suitable for implementing such process and for embodying these reagents and means.

A clearer perception of the invention will be achieved from the complementary description below, which refers to the drawings .

Brief Description of the Drawings Fig. 1 is a graph showing the quantity of GCH elimination during pregnancy.

Fig. 2 is a perspective view of an embodiment of an apparatus in conformity with the present invention for the purpose of implementing early pregnancy diagnosis; and Fig. 3 is a perspective view of another embodiment of an apparatus of the present invention. It must be clearly understood however that these drawings and the corresponding parts of the description are shown in merely illustrative manner relating to the object of the invention and that no limitation whatever should be construed thereby.

Description of Preferred Embodiments The apparatus shown in Fig. 2 comprises a support structure 8 with a rack or similar device 9 into which are fashioned recesses 10 for housing the various components of the apparatus, to wit: a dropper 11, a tube 12 holding the reagent and a tube 13 holding the diluting liquid. Support structure 8 is provided with a mirror 14 at its base, the lower part of the reagent tube 12 being reflected from said mirror. A screen 15 preventing interfering dispersion of the light and to the contrar favoring its concentration into the mirror and thus facilitating reading the reaction closes the rear of support structure 8.

As regards the embodiment shown in Fig. 3, the i_.ck or similar device 16 only holds two components, to wit: a reagent tube 17 and a tube 18 containing the diluting liquid, the latter being of such design that the volume of urine being examined may be drawn up as in a dropper.

Operation of the apparatus of the invention is as follows: Having collected the first urine of the day (possibly storing it in a sealed bottle away from heat), the diagnostician takes a predetermined quantity of drops by means of dropper 11 and introduces them into tube 12, which contains the reagent; then the diluting liquid is introduced — which advantageously may be distilled water — from tube 13.

After sealing tube 12 and shaking same well, it is put back on support 10 and let to rest on a plane surface, free from any vibration and heat, avoiding any handling in the course of the subsequent two hours of standing; the same care must be exerted at the end of the two hour interval. The diagnostician will then look into mirror 14 to see the bottom of tube 12.

If the image is a solid brown-yellow liquid, then there was agglutination reaction, and therefore the test is negative and there is no pregnancy.

If the image appearing in mirror 14 is either urine of unchanged color and turbidity (which would be the case of the substrate of the antigen GCH being made of a synthetic polymer) , or else a regular, dark-brown ring of variable dimensions (this is the case of the antigen GCH substrate consisting of erythrocyte then the agglutination reaction was inhibited by the presence of chorionic gonadotropin in the tested urine; the test is -positive and there is pregnancy.

If the equipment used is the one shown in Fig. 3, the diagnostician will first introduce the dilution liquid from tube 18 into the reagent tube 17, then will draw up the urine by means of tube 18 to introduce the urine into tube 17. The sequence of operations then will be the same as described in regard to Fig. 2. Indeed, the test being reversible during the initial half-hour, the sequence of addition of urine and of dilution liquid will not affect the end results.

It follows from the above description that regardless of the implementing means, or of the embodiments and particular modes , a new and novel process and a modified reagent will be obtained for early pregnancy diagnosis. Furthermore, new means for implementing this process and utilizing this reagent are provided, which, with respect to previously known processes, reagents and means, offer significant advantages that were clearly described above and which further advantageously allow extending their applications without requiring adaptation.

For example, they allow easy and rapid detection of chorionic gonadotropin in the urine of males afflicted with testicular tumors of the teratoma and epithelioma types.

As shown by the above, the invention will in no manner be limited to the embodiment modes , to the implementation and application that were described in explicit manner; on the contrary, it covers all variations that may occur to the expert in the matter without thereby leaving the domain or significance of the present invention.

Claims (12)

WHAT IS CLAIMED IS; ^

1. A process for early diagnosis of pregnancy in mammals for whom pregnancy causes elimination of chorionic gonadotropin of placental origin in the urine comprising: combining a predetermined quantity and dilution of urine from the subject being tested with a reagent comprising a substrate of erythrocytes or synthetic polymer suitable for use as such a substrate, on which are adsorbed chorionic gona-tropin endowed with antigen properties and a substance acting as an antiserum with respect to this hormone in a ratio so as to be sensitive to chorionic gonadotropin in the urine only when the latter is eliminated in the urine at a rate exceeding 1,500 I.U. of chorionic gonadotropin per liter of urine allowing the mixture to stand from one-half to two hours ; and inspecting the mixture for the occurrence of agglutination reaction or the inhibition thereof.

2. In a reagent for early pregnancy diagnosis, said reagent consisting essentially of a substrate which may consist of erythrocytes or of synthetic polymer suitable for use as such a substrate, said substrate having adsorbed thereon chorionic gonadotropin antigen and a substance acting as an antiserum with respect to said antigen, the improvement wherein the ratio of antigen to antiserum on said substrate is selected so as to endow said reagent with a selective sensitivity threshold as regards the amount of chorionic gonadotropin eliminated in the urine .

3. A reagent as defined in claim 2, wherein the¾ concentration ratio of the antibody in antiserum/antigen of said reagent is computed in such manner that the agglutination reaction will be entirely inhibited beyond the rate of 1,500 I.U. of chorionic gonadotropin per liter of urine.

4. A reagent as defined in claim 3, wherein the antibody concentration of the antiserum is increased by increasing the antiserum volume.

5. A reagent as defined in claim 3, wherein a selection is made from the various antisera provided by the various sources so as to obtain the highest antibody concentration sera.

6. A reagent as defined in claim 3, wherein the denominator in the antibody concentration ratio of antiserum/ antigen is made to vary by changing the amount of substrate on which the antigen is adsorbed.

7. A reagent as defined in claim 3, wherein the amount of antigen adsorbed on the substrate is controlled to control said ratio. the of Claim 1 ,

8. Apparatus for implementing a process/ for early pregnancy diagnosis comprising: a tube or similar device for holding a reagent; a measuring means for a given volume of urine to be tested; a tube or similar device for containing a dilution liquid; support for holding said tubes and measuring means; a mirror mounted on said support and positioned so as to allow inspection of the bottoms of the tubes or similar devices; and a screen favoring viewing the reaction in the reaction tube or similar device by concentrating its light toward said mirror.

9. Apparatus as defined in claim 8, wherein said^ V' tube or similar device for holding the dilution liquid is designed simultaneously to be used as said measuring means for a given volume of the urine being examined.

10. Apparatus as defined in claim 8, wherein the tube or similar device for holding a reagent has therein a predetermined quantity of a reagent so modified that when the urine being examined contains less than 1,500 I.U. of chorionic gonadotropin per liter, there will occur an agglutination in the form of a clear and homogeneous precipitate, and that when the urine being examined contains more than 1,500 I0U. of GCH per liter, the agglutination reaction will not occur.

11. Apparatus as defined in claim 8, further including reference images of the two likely reactions associated with the mirror reflecting the image from the diagnostic reaction tube, said reference images being used as standards for the image actually obtained and reflected from the mirror.

12. Apparatus as defined in claim 8, further including image fixing means relating to the reaction in the tube associated with said support. ADVOCATE, PATENT ATTORNEY P. O. B. 39251 TEL-AVIV, ISRAEL