CA1060794A - Composition and method for the determination of pregnancy - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A novel composition is provided for determining whether a female animal is pregnant. The solution consists essentially of chorionic gonado-tropin, preferably human chorionic gonodetropin (herein referred to as HCG) coupled with red blood cells of avian or camel blood by means of gluteraldehyde as a coupling agent therebetween. The composition may be stabilized with gelatin, or with formaldehyde. Use of such composition permits the pregnancy test to be performed within about 15 minutes.

Description

m e present invention Ielat~tes to a serodiagncfstic aomFosition for a method for the determdnation of pregnsncy.
In ~anafdian Patefnt No. 981,581 issued 13 January, 1976 to ~afa T,~fh~fratories Ltd., thfere is descri~ed and claimed a oamposition for det2r-mining whether or not a female is pregnant. 'I'hat oom~osition consisted essentially of human chorionic gofnadotropinf_oupled to red blood cells by - m.eans of gluteraldehyde as coupling agent therebetween.
Moreover, tha~ specification also described a meth~d for the determination of pregnancy which comprises admixing the aforesaid sero-diagnostic compositions with ~n anti HCG serum and a body fluid of a wcnansuspected to be pregnant.
In the event that the womar is pregnant, i.e., the HCG is present in the body fluid, the anti HCG serum wqll react with the free HCG and no reaction will occur bet~een the sensitized cells and the antiserum. Alter-natively, i.e., when the ~man is not pregnant:, the anti-serum will cause, - after 2 hours or un~er, certain conditions, i.e., wh~en gelatin is ~ corpora-ted into the aforesaid composition, even within 45 - 90 mdnutes, agglutina-tion of the serc~iagnostic c~position.
It isStatedin that specification that although the blood cells

2~ may be ob~ained from any animal, e.g., mice or even from human being~, the preferred cells are cbtained from sheep. It has nc,w unexpected1y been i found that when avian blood, especially turkey blood, or c~mel blood is used, the time required for the preformance of the test is tremendously reduced, i.e. to 15 - 20 minutes or even less.
The present invention thus in one broad aspect provides a c~mr position for determining whether or not a female anim 1 is pregnant, the ccmposition consisting essentially of chorionic gonadotropin (preferably human chorionic gonadotropin) (HCG) coupled with red blood cells of avian or camel blo3d by means of gluteraldehyde as coupling agent therebetween.

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A~s-- -` 1060794 - A preferred avian blood according to one variant of this aspect is turkey blood.
By another variant, the sGlution is buffered at a pH of between 5 - 8.5, preferably w~.ere the buffering solution is a boric acid-saline.
By yet another aspect, the solution also oontains stabilizing amount of gelatin, preferabl~ in an amount of 0.1% on a weight to vDlume ` basis.
By yet another variant, the solution also contains fornaldehyde, added after said coupling, in an amount sufficient to enhance the stability of said com~asition.
By another aspect, the compositions describel above are provided in combination with anti-chorionic g~nadotropin serum, preferably with anti-humJn gonadotropin serum.
By another aspect of t;his invention, a process is provided for -~ preparing the pregnancy-determining ocmposition described above, the pro-cess comprising muxing red bloodcells of avian or camel blood, and chorionic gonadotropin,preferably human chorionic gonadot~opin in a solution of gluteraldehyde, whereby the gluteraldehyde causes coupling of the (human) chorionic gonadotrop m to the blood cells.
~ By a variant thereof, the solution is buffered at a pH between 5 and 8.5, preferably with a boric acid-saline solution.
By another variant, the process includes thR subsequent step of r further treating the co~position with a rformaldehyde solution.
By yet another variant, the process includes the additional step of further treating the oomposition with gelatin.
If the cY~position has been washed with a borate saline solution, it may be stored for quite a long period. That period may be further lengthened by the addition of certain materials, e.g., gelatin. Such material is added, if at all, in an D unt of up to 1% ~eight/vDlume, preferably 0.05-0.2%.

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~ In order further to increase the stability of the serodiagnostic `- composition according to aspects of the present invention, the sensitized blood cells may be, after having been washed with the borate solution, further treated with gluteraldehyde or with formaldehyde.
Small amounts of the reagent according to aspects of the present invention and of the bcdy fluid are sufficient in order to perform the - method taught herein.
As body fluid there may be utilized blood, serum, urine, etc.
Preferably freshly filtered urineis utilized.

~ 10 Whereas in a preferred embodiment of the present procedure, `:' ' ~:

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` 1060794 the method is performed in a qolution adva~tageously in a buffer solution of pH 5 - 8,5, it may also be performed'in another form. Thus the sero-diagnostic composition according to aspects of the`present invention may be pressed into a tablet, be'impregnated'upon a paper strip or the like.
The so-stored'composition may then be brought, in a suitable manner, in contact with the anti HCG serum and the'body fluid of the woman.
; It is readily understood that the amounts of the various parts of the serodiagnostic composition have to be so chosen that the HCG is properly connected to the coated`red cells. It is not desirable to `' lO utilize a large excess of a~y of the'various parts of the composition.
-- Under certain circumstances, i.e.,'when both the sensitized ' cells and the anti HCG serum are lyophilised they can be mixed together and stored in this form. This admixture is also within the scope of ' another aspect of the present invention.
For performing the'method-provided herein, any known anti HCG
` serum may be utilized, as long as it is sufficiently specific for the HCG
utilized. However, advantageously an anti HCG serum as prepared by the method described'below and developed`in connection with the composition ` of aspects of the'present invention should be utilized. The amount of anti HCG serum to be utilized'is to be ascertained'for each serum as will ii be shown hereinafter. `
The'present invention in its various aspects will now be illus-trated with reference to the'accompanying examples`without being limited by them.
`'EXAMPLE_l .... .. .. . . . . . ..
A.-Preparation-of-absorbed rabbit-anti-HCG serum a. 5 mg of a HCG preparation having a potency of 2800 - 3000 U/mg : were dissolved in 5 ml of phosphate buffer of pH 7.1. The solution obtained was homogenized with an equal volume of complete Freund's adjuvant.

The'phosphate buffer'comprised:

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Na2HPO4 anhydride 16.06 g NaH2PO4-2H2o 5.85 g H2O up to 2000 ml The'above potency of the`HCG preparation is by no means critical for the success of the'immunization,procedure.
It is readily understood that any other suitable buffer solution may be utilized'as long as it fulfills the requirements. Th~ pH range may be varied between 6.5 - 8.5.
b. 0.1 ml and 0~5 ml of the'above homogenate were injected simul-taneously into the foot pad and intramuscularly respectively, into arabbit weighing 2.5`- 3.0 kg'once a week'for three consecutive weeks.

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,'~ c. One week after the'last set of injections the rabbits were bled and the serum was separated by centrifugation and stored in a refrigera-tor.

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d. Non-specific antibodies were adsorbed Erom the serum by mi~ing equal volumes of the serum and of normal human serum (not concaining HCG).
The mixture obtained is called "absorbed anti HCG serum".
B. Preparation of anti-HCG serum of high degree of purity a. 3 mg of HCG having the same potency as in Example lA above were ~` dissolved in a phosphate buffer of pH 7.1 prepared as described above and centrifuged for one hour at 15,000 RPM.
b. The supernatant obtained was added to and mixed with 7.5 ml of absorbed anti-HCG serum which had also been previously centrifuged for one 10 hour at 15,000 RPM, and 3.4 ml of borate saline was added and mixed.
The concentration of HCG and the volume of anti-HCG may vary accord-ing to the specific batch of the anti-HCG utilized.
The concentration is determined by preparing a precipitation curve , ~ and then selecting the optimum concentration.
The borate saline was prepared as follows:
i 0.85 sodium chloride ii H3B03 12.37 g ~i NaCH 0.52 g ;; NaCl 8.00 g H20 up to 1000 ml Each 97 ml of i were admixed with 3 ml of ii.

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c. The mixture obtained was incubated for 30 min. at 37C and then left ` overnight in the refrigerator~
d. The precipitate which formed was then washed 3 times with the above borate saline and finally suspended in 5 ml of borate saline.
e. 5 ml of the suspension were homogenized with 5.0 ml of complete Freund's ad~uvant.
` The homogenate obtained contains HCG which has been purified by specially binding it to anti-HCG. This preparation was then used for immuni-zing rabbits (according to Ab and Ac above) for obtaining anti-HCG antibodies of high degree of purity.
f. The antiserum was diluted 1:2 in normal human serum (not containing HCG) and diluted with borate saline to a suitable concentration and finally lyophilized.
E~YPLE 2 Preparation of the serodiagnostic composition a. Method A
- 1 ml of 2.5~ of glutaraldehyde solution in a phosphate buffer of pH 7-7.2 was added under constant stirring to the following mixture:
0.15 M-0.5 M phosphate buffer pH 7-7.2 10 ml ~ 20 Packed turkey red blood cells 0.4 ml - HCG solution (having the same potency as in Example lA in a phos-phate buffer of pH 7.1 having a concentration of 12 mg/ml 0.5 ml.) Stirring was continued for one hour. The preparation was then washed three times with borate saline, prepared as described in Example lB.
Following the washings of the HCG sensitized cells with borate-saline, the erythrocytes were re-suspended.

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~` 1060794 in phosphate buffer 0.15 M, pH 7,2. 'rhe volume oE buffer was the same as that used in the conjugatlon of HCG to the erythrocytes. While the suspension was gently stirred 1 ml solution of the same glutaraldehyde solution utilized - above before was added to the suspension.
- The suspension was kept under gentle and continuous stirring for 1 hour at room temperature followed by 3 days stirring in the cold room (4C).
After centrifugation (1500 rpm for 10 minutes) the supernatant was discarded and the cells were washed 4 times with borate salihe as described above. After the last washing with borate saline the volume of the packed ' 10 cells was measured and the cells were washed 4 times with 10 volumes of bi-`` distilled water each time.
Finally the sensitized erythrocytes .~ere re-suspended in 10 volumes of PBS containing 0.1% NaN3 and 0.1% gelatin which was prepared as follows:
17.5 ml ~a%HP04 0.15 M +
32.5 ml KH2P04 0.15 M +
50 ml saline (0.85 % NaCl) were admixed together and 100 mg of NaN3 were dissolved in the above solution:
Two ml of gelatin 5% in saline [5 g gelatin in 100 ml saline were melted and sterilized in an autoclave at 121.6C (2.0 atmospheres) for 30 minutes] were adoed to 98 ml of the PBS solution described above.
Next day the suspension of HCG-sensitized cells was centrifuged, ,, the supernatant discarded and the packed ce]ls were re-suspended in the same volume of ' '' ' `` -: ~ ~ .:
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PBS contailling 0~l% NaN3 ~nd 0.1~ ge]atin. lO volumes of the diluent per each volume of pac~ed cells. The obtained suspension represents the stock suspension of sensitized erythrocytes which was kept under refrigeration (2 to 8C).
In another case, following the washings of the HCG sensitized cells with borate saline the cells were treated as follows: ¦
The erythrocytes weré re-suspended in saline (NaCl 0.85%) to obtain a 8% suspension (based on packed cell volume).
l~hile the suspension was under gentle and continuous stirring an equal volume of a neutralized formaldehyde solution 1:12 in saline 3.3 g per 100 ml solution was added to the suspension. The mixture was kept under gentle stirring for 3 days in the cold room (N 4C).
The neutralized formaldehyde solution was prepared as follows:
The commercial formaldehyde solution of 36-37 g per 100 g (40 g per 100 ml solution) was first neutralized with 1 M NaOH (glass electrode) and the neutralized formaldehyde solution was diluted with saline 1:12 ~1 ml ` neutralized formaldehyde solution + 11 ml saline).
' After centrifugation (15000rpm for 10 minutes) the supernatant was discarded and the cells were washed 4 times with 10 volumes of bi-distilled , 20 water each time.--The cells were re-suspended in 10 volumes of PBS containing 0.1%
NaN3 and 0.1% gelatin, prepared as described above.
The following day the suspension of HCG sensitized .

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cells waj cclltrifuged, the superrultant discarded and the packed cells were re-suspended in the same volume of P~S containing 0.1% NaN3, and 0.1% gela-tin (10 volumes of the diluent to 1 volume of packed cells).
The method may be varied to a large extent. Thus the pH of the phosphate buffer can vary between 5-8.5; the HCG solution can have a concen- !
: tration of 4-12 mg/ml and the glutaraldehyde a concentration of 1-5%.
The working suspension to be used in the test was prepared before ~ distribution to the customer as follows:
: To 1 ml of the stock suspension, 25 ml of PBS containing 0.1% NaN3 and 0.1% gelatin were added. The working suspension thus obtained was dis-tributed in penicillin Wals for 10 or 20 tests (0.2 ml P ) i h a x cess of 20% ~2.4 ml for 10 tests and 4.8 ml for 20 tests).
Alternatively to 1 ml of the stock suspension, 25 rnl of borate saline containing 0.1% NaN3 and 0.1% gelatin may be added.
b. Method B
1 ml of 2.5% glutaraldehyde solution in phosphate buffer pH 7.1 : were added under constant stirring to the following mixture:
Borate Buffer pH 8 (see ii, Example 2) 10 ml Packed Turkey Red blood cells 0.4 ml HCG (potency as in Example lA)solution in phosphate.buffer pH 7.1 12 mg/ml 0.5 ml ' '' ` ' '' ,' ' ' ' `
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` 1060794 Stirring continued for one hour. The preparation was washed 3 times with ` borate saline prepared as described in Example lB and then stored in a re-frigerator.

Example 2, both method A and method B were repeated, however using camel red blood cells rather than turkey red blood cells. In all other respects the preparation is identical.
With both the serodia~nostic composition of Example 2 and the sero-diagnostic composition of this example pregnancy tests as described below were carried out with results obtained within 15 to 20 minutes.

Standardization of anti-HCG serum . Two fold dilutions in (borate saline) of anti-HCG serum were pre-pared in suitable test tubes~ The volume of the serum in each tube was 0.1 ml. and 0.25 ml of HCG coated red cells prepared as described in Example 2 were added to each test tube. The concentration of the red cells was approx.
0.4% of packed cells. The tubes were shaken and left to stand at room tem-perature for about 3 hours. At the end point the results were read and the concentration of the highest dilution of the serum which still agglutinated ~' 20 the red cells was determined. This concentration is the end point of the titration tnd i8 defined as "one hett~gglutination unit".

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For routine pregnancy tests a suitable concentration of serum is used which may vary from 2-16 hemagglutination units the optimum being usually 4 hemagglutination units (a serum 4 times more concentrated than the end point).

Performance of the pre~nancy test (Method 1) The following reagents were mixed in a test tube or any other suitable container:
0.1 ml of anti-HCG serum of suitable concentration which was deter-mined as described in Example 4;
0.1 ml of filtered urine of the woman the pregnancy of whom is to be determined; and 0.25 ml of sensitized turkey red blood cells, the concentration of the packed cells being 0.4%.
The mixture was shaken and left to stand for 15-20 minutes. If HCG is present in the urine, i.e., the woman is pregnant, it combines with the anti-HCG and therefore no agglutination occurs. If the woman is not preg-nant so that there is no HCG in the urine, the anti-HCG is free to aggluti-nate the turkey red blood cells, as shown by controls to which 0.1 ml borate saline has been added instead of the filtered urine, and this agglutination ?
indicates non pregnancy.
As indicated above, the test is completed within 15-20 minutes.
In the case of the use of sheep red blood cells instead of turkey red blood cells in ., . .. . .... . - , . -. :

ot!l~rwise the same composition as described above, the time required for the test is 1 hour. In the case of the use of sheep red blood cells and a composition without gelatin, in addition to the decreased storage life of such composition, as described Phove, the further disadvantage is that the time required for the test is 3 hours.
In the case of the use of the camel red blood cell composition of Example 3, in the place of thé turkey red blood cell composition of Example 2, the time required for the test is 15-20 minutes, i.e., the same as with tur-~ key red blood cells.
`~ 10 EXAMPLE 6 Performance of the pregnancy test (Method II) The anti-HCG serum was lyophilized in vials each containing anti-~ serum sufficient for 25 tests. The powder was dissolved by adding to the con-tents of one vial S.O ml of the sensitized turkey red blood cells the concen-tration being 0.4% of the packed cells.
- The reagent was subdivided in amounts of 0.2 ml into test tubes or other suitable containers. To each test tube or container 0.1 ml of filtered urine was added.
The mixtures were shaken and the results were read as in Example 5.
The results of clinical tests performed with preparations manu-factured according to~the above method are given in the following tables:
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CLINICAL RESULTS OBTAINED WITH THE PREPARATION OF EX~PI.E 5: conc. of serum 2-4-8 hemagglutination units (HU) 0.25 cc 0.4~ red blood cells coated with HCG 12 mg/m. as compared to results obtained with a commercial preparation (CM) Se No 8HU 4HU 2HU CM

3 + + + +

4 + + + +
+ + + +

7 + + + +

g + + + r - 1 1 -- _ _ _ 12 + +

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x = pregnant - = non-pregnant -, : : : . . --- --~' - .-' - : - . : -.

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TABLE II
Cl.INICAL RESULTS OBTAINED WITH THE PREPARATIO~ OF EX~'IPLE 4 (conc. of seru~ 4HU) 0.25 cc 0.4~ sheep red blood cells sensitized with HCG
4 mg/ml as compared to results obtained with a commercial preparation (CM).
Ser.
No. 4HU CM
-- - _ .~ ~ 2 _ _ . 3 . 4 +
6 -- _ . 7 +
; 8 +
g ,, 1 0 11 + +

' 20 + = pregnant - ~ no=-pregnanc :

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TABLE III
CLINICAL RESULTS OBTAINED WITH THE PREPARATION OF EXAMPLE 3: (4 HU units) as compared with a commercial preparation (CM) and with the Quick Hyperhaemia test (QHT) _ ,, + ? +

+ +

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Claims (20)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A composition for determining whether or not a female animal is pregnant, said composition consisting essentially of chorionic gonado-tropin (HCG) of the same type of animal or said female coupled with red blood cells of avian or camel blood by means of gluteraldehyde as coupling agent therebetween.

2. A composition according to claim 1 wherein the avian blood is turkey blood.

3. Composition according to claim 1 suspended in a solution buffered at a pH of between 5 - 8.5.

4. Composition according to claim 3 wherein said buffering solu-tion is boric acid-saline.

5. Composition according to claims 1, 2 or 3 wherein said solu-tion also contains gelatin in an amount sufficient to stabilize said com-position.

6. Composition according to claims 1, 2 or 3 wherein said solu-tion also contains gelatin, said gelatin being present in an amount of 0.1%
on a weight to volume basis which is sufficient to stabilize said composi-tion.

7. The composition of claims 1, 2 or 3 wherein said solution also contains formaldehyde, added after said coupling, in an amount sufficient to enhance the stability of said composition.

8. The composition of claims 1, 2 or 3 wherein the chorionic gonadotropin is human chorionic gonadotropin.

9. A pregnancy-determining composition comprising, in combina-tion, the composition according to claims 1, 2 or 3 and anti chorionic gonadotropin serum.

10. A pregnancy-determining composition comprising, in combina-tion, the composition according to claims 1, 2 or 3 and anti chorionic gona-dotropin serum and, in addition, gelatin in an amount sufficient to stabil-ize said composition

11. A pregnancy-determining composition comprising, in combina-tion, the composition according to claims 1, 2 or 3 and anti chorionic gona-dotropin serum and, in addition, gelatin in an amount sufficient to stabil-ize said composition and, in addition formaldehyde added after said coupling in an amount sufficient to enhance the stability of said composition.

12. A pregnancy-determining composition comprising, in combina-tion, the composition according to claims 1, 2 or 3 in which the chorionic gonadotropin is human chorionic gonadotropin, and anti-human chorionic gonadotropin.

13. A pregnancy-determining composition comprising, in combina-tion, the composition according to claims 1, 2 or 3 in which the chorionic gonadotropin is human chorionic gonadotropin, and anti-human chorionic gonadotropin and, in addition, gelatin, in an amount sufficient to stabilize said composition.

14. A pregnancy-determining composition comprising, in combina-tion, the composition according to claims 1, 2 or 3 in which the chorionic gonadotropin is human chorionic gonadotropin, and anti-human chorionic gonadotropin and, in addition, formaldehyde, added after said coupling, in an amount sufficient to enhance the stability of said composition.

15. Process for producing the composition according to claim 1 which comprises mixing red blood cells of avian or camel blood, chorionic gonadotropin in a solution of gluteraldehyde, whereby said gluteraldehyde causes coupling of said chorionic gonadotropin to said blood cells.

16. Process according to claim 15 wherein the chorionic gonado-tropin is human chorionic gonadotropin.

17. Process according to claims 15 or 16 including the step of buffering said solution at a pH between 5 and 8.5.

18. Process according to claims 15 or 16 including the step of buffering said solution at a pH between 5 and 8.5 with a solution of boric acid-saline solution.

19. Process according to claims 15 or 16 including the subsequent step of further treating the composition with a formaldehyde solution.

20. Process according to claims 15 or 16 including the additional step of treating said solution with gelatin.