Method for the detection of pregnancy disorders using a radioactive iodine-labelled antigen represented by the protein pp13

י’' ' ..' / (The present invention relates to method for the detection of pregnancy disorders. More particularly, the Invention \ relates to a ; /; method for detection of placental damages at a

. - ..'. '. י'*.,/. . . ' .

,.'/.״ *relatively early stage of pregnancy raE invention׳¹5'BACKGR0UMP(y ./.״

- , י' י , ** .>.!- ׳יי. י*,'^,,”' י . . '* . * . '. ^v

25% As known, highrisk pregnancies constitute about 10 to י' /:י, gfpregnancies. Among the high risk pregnancies disorders the י following can be mentioned: diabetes, kidney diseases (such as . . clonic pye renal insufficiency); heart disease (such as primary pulmohary) .

,: י‘' . ־י‘/. ־ 4' 'י“‘, ' /י'.* J 'י'.־ ' ’ י' ־,. . \ י .F hypertension;,. The known methods for controlling the pregnancy

I '.,/. ' ,. ' - ’ . ־ ־ . ^:^־. ׳ . ,^Tי '^J . '־ י'־ I־' ’ '⁴’ progress, have the disadvantage that.they detect the status of .' .. pregnancy disorders at a stage when the clinical signs and symptoms

' ' / ' ׳ / /. ' ' : ?//'־.׳יי ' י ^{1 ::}: , ׳׳ . ¹ ’ / ; are already apparent. .

׳־ ׳ //.-. ׳׳ . .? ־. ₄ .׳׳ ^!״' ׳ י. יי י .׳<׳ , ־: ״ ׳ . ¹ ׳,.׳ . יי.־' ⁷י;

. There are several hormone assays suggested to give an ^in&cation impending fetal death. lhe tests most widely iised are: urine estriol< urine-total estrogens, serum unconjugated estriol and serum placental lactogen* As known* estriol is an estrogenic ׳/ ׳ ־'^, . ; . r* ,,. -‘״ א ./ ־, ; J.'; 7׳ ץ'׳ \ ' ‘ ',χ,יי־' ד.’^,?״ -. ' ־'⁷ ־ ־ r ,².⁰ hy the/placenta from precursors derived from/ fetal adrenal cortex and fetal liver* ־ The conjugated form of ^striol is excreted in maternal urine. Serum estriol can be measured either as total estriol or as unconjugated estriol, it \ usually is measured , as unconjugated estriol in order to exclude ־ * ’' ' .Ϊ , ן , . זי ' . יי . ‘ . . ך ־ ' _L ; ‘.

י ־ . י .י . . .י - 2 י ' י־ ' . ^: 'י': 75 .maternal contribution to the conjugated fraction. Urine estriol

J ' cm since estriol normally constitutes about 90% of urine total estrogens. _t

Estriol can be detected by. immunoassay as early as the ninth . 5 week of gestation. Thereafter, estriol values slowly hut steadily increase until the last trimester, when there is a more pronounced increase. Clinical use of, estriol measurement is based on the fact that severe acute abnormality of the fetoplacental unit, such as a . ¹ / ' ־- . * ן . .'י - ’ ^י ’ . ^; '׳ י ,. י־ 1 . ־ - * ' -. < ‘Λ , ;׳- - . י ־ . ¹ . 7 , _ל, ’ dead or dying placehta, is manifested either by failure of the estriol level to continue rising or by a sudden marked and sustained decrease in the estr 101 level. A very recent report (M. Scharf et.al. J.Otetet. Gynec,reprod. Biol., 17 365-75,:1984) concludes .: that in view of the low correlation between patients with abnormal . serum free estriol as the antepartum pathological test, the estriol : 15.; measurement can .not be considered a reliable predicting tool to ' , ' * ‘'' . , 1 . .' ׳» ' ׳.־ / . .־ י י' * - ’ Λ \ 'ר‘ ׳ י . . י יי .’׳.* . -.,. .י .. .

. estimate the actual postpartum state in the, pregnancy disorders.

Urine total estrogen was the first test used, since total estrogM can,be assayed by standard clinical techniques. However, ׳ y urine» glucose, falsely increases the results and certain other 20 substances such as urobilinogen also may interfere. On the other hand, maternal hypertension, preeclampsia,: severe anemia and \?׳;''׳i11^idred_:-raua.'־func^^ can decrease urine estrogen or estriol ’*.' . ./.f''. '>׳.. . '.'Λ . ,“ ^,‘יי’ *'Λ ׳ י' * .י * י . י י secretion considerably. Decrease in the contents, may also occur to variable degree in a number of fetuses with severe congenital

¾¾/ -. 3 -

׳ ’ . ־ י ^{1 1} י י . י , K‘ *

(־ 1. ‘. . . ׳ «* .־,*־».' י.

anomalies« It was also reported that continued bed-rest to the pregnant woman, caused an Increase in the estriol excretion values of about 20 to 30% over the levels determined from ambulatory persons. ל^גי$£<?.. »»!*.a ׳.,׳,. י.

Because of the problems associated with collection of urine or serum estriol specimens and interpretation of the values, as well as the disturbing number of false positive and negative test results, most of the clinical people refrain from correlating these measurements with placental damages. Moreover, all the previous methods did not reveal the pregnancy disorders at an early stage of their appearance, but only when the particular disease was already existent.

There are some prior art references which deal with the measurement or identification of placental protein. Thus according to H.T. Salem et al (British Journal of Obstetrics and Gynaecology, 88, April 1981, pp. 371-4) the circulating levels of placental protein PP5, placentai lactogen (hPL) and pregnancy-specific Bj^-glycoprotein (SPp were measured in pregnant women at the 16th week of pregnancy and the levels were correlated to fetal outcome in late pregnancy.

In another paper by H. Bohn entitled: Systematic identification of specific Oncoplacental proteins [Oncodevelopmental Biol. Med. 1983, 4.(15) pp 343-50] there is a discussion on some biologically active׳ compounds which are specific to the placenta. These compounds which are in their most part proteins, may have the function of hormones, enzymes, proenzymes, inhibitors, or receptors. Their detection and measurement in the circulation is ־3 a ־ used to diagnose pregnancy. Proteins immunochemically identical or related to the human placental-specific proteins, which were found in extracts from placentas of women were SP, , pp_r pp pp dd t+1' 5׳10 ׳ ^{and ρρ}χ2* ^{Tt ls} specif1cally mentloned (H.Born, Systematic identification of specific oncoplacental proteins, chapter 4, p.ei, in the book Oncodevelopmental markers, Academic Press 1983) that measurement of PP₁₂ appeared to be of no value in monitoring pregnancy.

SUMMARYQF THEINDENTION

It is an object of the present invention to provide a simple method for the detection of pregnancy-asociated disorders at a very early stage of their appearance. It is another object of the present invention, to provide a simple method for the detection of pregnancy disorders, said method being of a very high sensitivity. It is yet another object of the present invention to provide a simpie method for the detection of pregnancy disorders, said method being not influenced by other extraneous factors related thereto. The present invention is based on the approach by which antigenic compounds are released from a

- 78237/4 pathological involved tissue into body fluids, using an immunological specific antigen which is derived from human placenta, and can be subsequently determined.

Thus the invention consists of a method for determining pregnancy disorders by analysing a biological fluid sample, which comprises the steps of:

(a) labelling by radioactive iodine an antigen consisting of a non-hormonal human-derived placental soluble protein known as PP-13, characterized by the following physicochemical properties: (1) electrophoretic mobility, in the range specific for albumen; (2) isoelectric point, in the range of between 4.6 to 4.9; (3) molecular weight, below 35,000 Daltons; and (4) carbohydrate content, below 1%;

(b) incubating said biological fluid sample with the labelled antigen׳ and its corresponding highly purified antibody which possesses a highly immunologic specificity towards the purified placenta antigen (hereinafter referred to as first antibody);

(c) reacting the product obtained in step (b) with a second antibody having the property to precipitate a complex (hereinafter referred to as second antibody); determining the radioactivity in said complex, and (e) determining whether placental damage has occurred based on the level of radioactivity in said complex.

a ־

It was surprisingly found that using the above procedure with the particular protein PP-13, it is possible to establish an early detection, of pregnancy-associated disorders in a pregnant woman. The invention was found particularly useful for the detection of the following specific pregnancy-associated disorders: Severe preeclampsia, Intra-Uterine Growth Retardation (IUGR) and preterm delivery.

To-day there is no other comparable test enabling the e.ftrly diagnosis of placental damage and/or insufficiency placenta leading to pregnancy disorders but only after their existence in an advanced stage of placenta illness.

A soluble protein possessing the above physico-chemical properties, found to be suitable for the present invention, was discovered in the last few years (by Bohn, Hans et al) and named PP13. This protein is •^; ׳ .א יי־ .־ א ; /. י - ., .5 א. .?>/ ' -//' י

Q.' ' ־־ ' )־ י ׳ \ . Ζ ' ־ ־ '־ ־־ ¹י ־ ' . ' ׳ ־־ ' descriJ^ lri\ the W (assigned to Behringwerke AkUengeselschaft).As mentioned in the .

־ . ׳׳/ . . ₎ ' יי׳ ' . א/ patent specification, this protein could be useful far the diagnostic purpose of tumorsdetection of trophoblastic character.

The discoveryaccording to the present invention, to utilize a placental protein for the prediction of occurence of pregnancy , .*- ־ * ' I ' * . ’ . , '^v י -♦ .. ,. . . , ־ ,7 ׳ י י י '. , disorders is quite surprising, ih view of prior art statements which discuss various placental proteins, stipulating that they are of no value for this purpose (British Journal of Obstetrics and (^aecology,-December 1984^ The inventor

,. . carri^ cwt'a number of experiments with various placental proteins • which do not possess ail the above four physico-chemical properties and f<^ indeed that'there are !my placental indeed ,not suitable for the present invention although they possess one or bro of the Thus» for instance KM'.Has a

־. molecular weight of>35,000 (i.e., as required by the present ) invention) but Its carbohydrate content is 2.4% compared, with below

י י .א^,'׳. י.'.^,:/ י״. . ׳ .’>׳ . ׳ ׳./׳;.א.'׳־' י^,־ , .! 1% required by the present invention and accordingly was found to .

’.־; be unsuitable. :, ''' 2 ׳' ;20 * Another placental protein, named PP*9 was described and characterized by H.Bohn et al (New soluble placental tissue proteins; Immunology of Human Placental Proteins, Placental Supplement 4, 1982 Praeger Riblishers). Its Isoelectric point is given to be .between

6.4-6.7 and carbohydrate content 5.5%, which are not according to \'-‘ . . ’ ־ ' ' * ’*, _{f L}. ;״.’.׳. . ‘ , י ' I . ' .,

־.¹ /א.'. ‘/./'.{ ז^,.*Ί.: ' ‘ ¹ .’׳. 'י:־.־' .. א י V׳; . Λ .

/י / ׳'אי/ / א1/>'^;א'.א . Γ ' Λ ' :'/ ' ί י . . =,׳. . . :-׳ .,, - y

J.Z;^;ZZ י'-''' ' ־. .'.Λ' '־ י‘-Z ^e י'׳'( '.י ד the present invention. The inventor has found that PP-9 is not . Z\' ' Z: <- .ל,.', V ././ ..'׳ ' :., '.;י/.' .>;.׳ .ג,׳ suitable for the method being of no value in the prediction of ¹ ' *>>'. .׳ .’-י'. “. .Z ’ ' .. י״/.;‘ . . ׳ ’;

occurence of pregnancy disorders. ;

> Z The inventor is not yet in a position to explain the

-j'5׳ theoretical aspects; why only a placental protein.possessing all the ׳ > /, . * ־ ־* ^ד .: ך. ״ Z* ׳ ' . »' ׳./'., י ׳'.> י י . יי' :\ ׳ ׳ י .'. ; , ־. .

above four physico-chemical properties, such as PP-13, is suitable for the present method/while other placental proteins are unsuitable. . However such theoretical explanations are beyond the .

. sa^e of the invention Z '׳. Z.

.י'־‘ , ’״׳׳'״ . Z Z Z -Z’ .׳־״,' ׳׳'׳ ׳.’־“. ׳ λ Amongthe biological fluid to be analyzed, for the antigen determinationaccordingtothe present invention, the following , shall be mentioned: blood, amniotic fluid or urine.

, PEsaagniCN cap toe preferred emvcdiments ׳ fii^t'6t^.gf41abelling. the.Er0tein,,by.racli0active iodine, is. !mown in .־die art by the term radioimmunoassay.As known, the radioimmunoassay is based on the canpetition of a known excess . > amount of radiolabelled antigen and. an unknown variable amount of, . . ׳׳ J¹ . * י . . . . ׳' '' ^וי ^ . ‘ ׳ .י^,.,- > ׳,« ׳ the unlabplled antigen, for binding sites of a constant amount of .

antibody. The specific particular method selected for labelling the antigen, ;is the so called solid phase antibody. This labelling was carried out according to the method of Marchalonis (Biochem.J.H3, ־ ״' z . * , . . ?',A.'. ’ . .' '* ־ ,.־^,”' ' -'־י ’ ' ' , . 1963 ). with Lactoperoxidase-fi₂O^. Due to the .,alkalinity of the '7. ־ , ׳ י ., ־'״- . 'Z Z י.^,,:., י ' mixture,־some phosphate buffer saline, was also added so that the

ί ,. י' ־ '־ ? י' : .י,.‘Τ Λ',;. •

ί 'י/ ־ לי י יי' . *'.>־.< רל-ליל

> <' יי' ל../:׳' . / '/ל Λ\:¾ ' -׳;׳׳־/'/.ל ילל;

i . י ' י. י י יי;,. ¹ יי י '״' י-

j . . . י .י. .* י י י *י י.. ₎ יי^, י. י *יל י. ^j י י. י יי^,.*.. ״*׳«״

I י' : .י׳ . ¹ 'ν- י' י י- . ' : >' י .׳ * '־ -י ' י ,י ^; ' Ο

ך ״׳ .’ .ל'^;לל׳.-:ל'.-? '7׳:?/>;^ . .;׳׳.-',.’יל.,:: י ־' . י ^:

* . ’ י . י י ’ י , , . /.- י׳ - . ץ ' ' mixture reached a neutral point . The vise of radioactive isotopes ' I י λ has enabled the immune reaction to be more easily observed, and thus ל j ¹ ’ ׳־י .:i \ 'י V י' ־ ' ‘ ־ ' ' י ׳ י‘ י'. '. י, ’ /; It is preferably to be υίΐΐΐ^ ώ the present i^ ־׳*',' ^k . Λ. ־ . ' . ., . . , ’ ן י׳',,. ’ . '..־ ’ . ׳י''. ל *,' .

the specific protein can be determined withgreat sensitivity.

' ., ,. . . ' .' . ' ’ יי ’ ' ^Γ| I , : ' ' . . 1 ' ’ . ' . I, _:t ’.

The radioactive iodine bounded by the antigen is counted, , , . ⁴ ' ' , 1 , F . 1' ' . . I 1< ־> ' ־ . ' ' . , | . _r ' ‘;

.׳ using aGamma Counter( such as Auto Gamma produced by Packard) and

/.״ &lt;'.,’.׳the yield thereto is calculated. The yield is calculated using . ,, \ ’.^,,.'“‘.יי ־. ' י - ' י' ’» ' ’ ייל. . . * ’ ־’׳. .' .

the foilowing formula:

'׳'/.,.י ־’ '. ,/; ־‘ &lt;.- , p .., ' י' ' : ,־ ' /.' ,

Yield = radioactivity measured by the peak of protein x 100 /י׳ -.,־ .׳/ל .,,/ל/ל/\י T^ ,radioacdvity ν . : ; , י^ ־׳

1&lt;י.יל.:.;..&lt;) ' ’ _ύ , ,,The most preferred concentrations of the antibody for .- .*לי'.⁴ ‘ ־׳ \ f ' ;.^rf - ־' - ;.* '/'*., . ‘‘ ' י ’ ' ' ‘ obtaining.;^ - AB) is between 1/10,000 to ;.

1/50,000. In this range it was discovered that there is a linear ’ . ' ^,ל׳ .;יו־ '’ . ^J '׳ —.'^1, ‘ ' י _J ' ,. '. . . . . , . ‘ ל ׳ . י' . ־’ ל correlation between the specific binding and antibody concentration. ;

; This spears in a,clear manner in Figure 1, wherein , the specific , , . '<׳_t'''י ’־ ־.’^1,.': ל'' י י. \ ' ־ . -־‘^,'-'-י. '״ ,15( binding (expressed in percentage) is given versus the concentration ׳/--. ׳ . ל-׳>־;/ ^r. ללי ?ל יל' /.׳״' ' ל^:־)יל י , / ; of the PP-13 protein. Based on the yield of theradioactive iodine.

ό ; . . . 1 ־ ’ ' ״♦׳־'. , ' ',.־’. ”. ׳ ’ ^! '4 . . . ־' A . ־ ־' . . ' '. . '.׳’ . '־A : ־. ' ' ' .

; the specific activity of the labelled antigen is calculated and י י ,. ' 4 י‘ ' י ¹ . ** .י ׳״ י '. , . ׳' י' י , ; r - . ’ ’*,׳.

subsequently this is correlated wi^ . In the second step, the labelled antigen obtained in stq? 20 (a) is correlated with the biological fluid (blood, amniotic fluid ׳ / ; ׳: : or urine) and a first antibody* This antibody was obtained from ל Lvyv.7£s^a9ron'־iof _:&lt;tte.placental'^,m diluted by a buff er ״-.;./(pii «4.7 ׳) consisting of Nafc (1 g/1) and TRIS - HC1 (0.02 mol/1).

'/ '־8 -י’ י' י . ^Λ'^: ..׳־ י /־/,; , . v 4 . ;4 V . . י ׳ . .In the third step, a secondantibody, attached to a solid : ., support, developed in the serum of a donkey, was admixed 'with the product of step (b) and the precipitated complex was separated by . centrifugation and washed with a Phosphate. Buffer Saline solution.

Finally, in the fourth step, the radioactivity was .'y. . determined in the washed complex by an Auto Gamma (produced by.

Packard)/ ’ /' , ’. ' . . ’' ' . ' - , ' ' ⁴ . . , ' 1 .׳ r י.^,'* *.’־, י י . ״' *׳ ‘ .

The above steps of the method will enable to obtain the . ;ν'¹'. . . ’ .'. י'''' .י. ' ' ' ' y'*'־ , \ . ., '.

following data: ;.׳.׳׳.׳ .,. . י. , .* ־. ׳ ׳;׳ ׳ . .->״'׳' .׳ ׳ .י י .“' ׳ ׳ ‘ '.־ '

1) 10 ^ז) The degree level of total radioactivity introduced in the . system’of the, experiment; : .׳ .- , / ׳ (2) The degree of.non-specific binding of the labelled antigen and . .* the second antibody; ..

' ; / (3) The! zero binding of the antigen and its specific antibody;.

(4) Calibration of ;the system by utilizing a series of known ‘ concentrations of the :unlabelled antigen.; ׳

In the attached Figure 2, it is presented the radioimmunoassay . /λ' 125.'.' ' י . י'.'' ' ' י. '. לי . ., ^v.' calibraUoncunreof the protein,correla ting the l radioactivity (expressed in counts per minute) versus the PP-13 protein concentration ( expressed in ng/ml).

/ The methodaccording to the present invention was applied ? to four groups of population samples: (1) adult׳ ' males; (2) adult . י' -י י*‘ . י־^,.' י ־ יי י י . י־ .י ' ..׳ .׳ ׳' י ^:.י . ,. ' a non-pregnant females; 3) -י) adult asymptomatic pregnant females;

¹ ’ ^χ. י־ '- ^s *- - ׳ . י י ’* ־* י ״ . י ' . . , . J;\׳ y.^.';U);.,ai^^ The same procedure, with exactly the same reagents and amounts thereof, was employed —¹¹ ._י \ . ' י. ' .« ' ' ־י H »'; ' ׳ . ₎ * . י . * ׳ , י f orallfourgroups and the results obtained are Illustrated in

3. In this Figure, there are correlated the cumulative distributiai in the sera for the above four groups

. . _ . ‘ ' . ¹ .. י'.'^, ' .

ver sustheconcentration of the PP-13 protein. From the graphs .y . .y Ϊ ,y' ;..y. ' ׳ .- .y\. ' ;

presented therein it can be noticed that the group of pregnant females differ from the groip of males as“well as the group of non-pregnantfemales. It can be,also noticed that, there is a substantially equalconcentration of protein for the adult males . and non-pregnant females at the same cumulative distribution.

‘ ‘ ' י ׳ .י’,.« -. ן׳'. , ' * ־ f . ־ .’ ־ S* . ' '

However, there is a sharp difference in the protein concentration ? between asymptomatic pregnant females and symptomatic pregnant females, this concentration being substantially higher in the .15 latter group than ih the former group particularly at a cumulative ' . distributicm of 'sibove 0,4, the gap being particularly sensible ! veien the protein concentration is above 0.7 ng/ml. The ' interpretation method is according to Siegel S (Non-parametric v statistics for the behavioural Sciences, Mc.Graw-Hill, 1956, New

'.20-, .' '״' ,.York)., ?,״ ־ - ^ז־ י ; ; In the following Table l are presented some statistical values concerning the distributional pattern of the PP-13 protein taken as antigen, determined with the method according to the *1_; :. /1 _.,־ ;. ר .,' - 10 ך.., ,λ,. ״י . י' . י.,.\ י .י .׳ . י . י ״ .׳!׳.?, י' י. י ׳.׳.,, ¹. present invention, for the above four groups of population samples* !'׳ ., _t, . .' ,. w . . ‘ .

^k . ־; . i , , . . ‘ ־ . .־ . . ' ׳ ’ . _§ The protein concentration in the blood samples tested was in the ¾-. ׳/ range of 0.3-2.4mg/ml. .־.'' /y'/^V TABLE 1, ^: '.׳../' . _ר . ., .־.&lt;' י י ' Statistics of the distribution of the PP-13 protein tested .in sera of males, nonpregnant females, healthy pregnant ’ ^feff|ales and nonhealthy pregnant fenales♦

Males Nonpregnant , Pregnant females .;..&lt; Healthy־. Nonhealthy

<td> 10 Number ‘ ; . . ׳. ’ .</td><td> 18</td><td> 48 /'</td><td> 92</td><td> 150</td>

<td> . Mean (ng/ml) ^:</td><td> ‘0.58</td><td> 0.61</td><td> ; 0.82</td><td> 0.95</td>

<td> . Standard deviation ' , : ' . . . .־&lt;!; ' *</td><td> 0.14 </td><td> 0.20 </td><td> 0.16</td><td> 0.34</td>

<td> ''. 1st. Quartile ׳ ' ־. ׳ y</td><td> 0.46</td><td> 0.48</td><td> . 0*71</td><td> 0.71</td>

<td> ״־ ¹ &gt;, .-. . .׳ &lt; . . y / : 2nd Quartile ־ :</td><td> 0.58</td><td> 0*56</td><td> 0.82</td><td> 0.90</td>

<td>¹⁵. 3rd Quartile</td><td> 0.67</td><td> 0.68 .</td><td> 0.90</td><td> 1.15</td>

Frpnrthe results presented in the above Table 1, it can. be noticed that the protein level is substantially the same in the ’ ' , '־, *ל,' ' - r ־ ' . ־¹יי , ‘ blood samples of males and non-pregnant females. The level of protein appears to be higher in pregnant females, where in about

75% ofthe pregnant females there is a protein level of above 0.71 ng/ml. This difference which appears in the Table is significant, ' from a statistical point of view .(P = 0.0035) using the Mann־’. V’ ,’ : , ; ' י' י *“״^f.

'Whiter also be noticed that in about 50% of

;׳.'.*’ ' . י׳ י ' , &lt; ' ״ ' . ' . ;״י' י..‘.,-

,. ־ . « ‘ . ‘ '. .. י - '־.*' ־ίί , ; ־ , . ' > nonhealthypregnant females (compared with about 28% of healthy pregnant femalesl. ^ 0.9 ng/ml.

׳. 7.;י^According' to another embodiment ׳of־ the present invention. , .^׳;Aaa' AAA׳ , A'a ־ ' 7 ;--.ג. .;7 < .7> 7.7 ..?י, A׳‘ theplacentalproteinpossesslngthe above mentioned fourphysico׳. ׳,' A ׳,.־ ׳ ׳' ’ * - ’ ‘ . '־ - '' 7' ־ ׳ ׳ י -A A .,. ' ’ .!י .

&lt; 5 chemical properties, such as PPAL3 can be utilized not only for .di.agnostic. hit also for monitoring the pregnancy development and ocairrehqrcf placental damage. The method can reveal any abnormal development due to medical treatment far a pregnant female such as &lt; diabetes, anemia, hypertension, rhenal, thyroid etc. Based on the 10 results, the physician will decide whether the pregnancy should be ceased,or to change the dose of the medicine, or even to change completely themedicine taken by the pregnant female.

7/.αΑ^; '7 A'A A ^,''-A -. -, A.' /7 7v Thus the pregnancy monitoring by the method according to the ' ' .״ :. r י’¹ י . ־. . ׳* ' ' . .* ' ־ ' . ‘ .'־ ’ ' . . ; ^; .' '.־ pr^smt ln^Uon will be of high importance, belng most appreciated / .׳ ' .י^,,A , , A ., ״' ' i j ,. ./׳ ’’י ’ . . .

«!5 j;y a person versed in the art. this is a most surprising finding,

A in view of prior statements which conclude that placental proteins could not be specific to pregnancy in view of the differences which

A ;•/•7 . exist between them. .'׳. ;. ־ '' ׳'>׳: A . F .' '. י ׳׳׳,' .. י .- ’י י '׳. ״ ׳ ' ,A .

. While the invention will be hereafter described in a detailed manner witii certain preferred embodiments and it will be understood that it is not Intended to limit the invention a t0:th®ise particular embodiments. On the contrary, it is intended to * cover other alternatives, modlfications or other ingredients1 ιώ may be included the < appended C^aijns, It should te unSerstood that are ? ' -X ' '.' >' '^״,; יH X* ' .J י , י. י י’ ,. /) 'י *. י 'י, י L X * .י ty way of example and far purposes ofillustrative discussion of : . .־. V- /.־ .> ' . . . - . .'. X . ' ' י .־ ‘ ־. ־ . X־ , ׳' . .

X. XXX ־..>-. , '.־. ,.'..׳.- _f ־, . ¹ ק, ׳ x/ tte procedure for carrying cut the method according to the present . ;5׳: ;indention, without being limited thereto./\ in the fallowing Examples and the source are as followss'X .

, .tot 'N0/2n7»3.\/ ־, 1 . . ' ' ־ , * , ‘ . A » i . > , .I . . . ,.

10X AntTP^ 'י ' X/ V'X ־’/ :. X׳ / ״. x^: .. ; ,.

'?Ί./ \\ X' /׳7 .׳':. /'.'/ x ־ . iodine ( I) produced by Israel Nuclear Center / in a solution, at Sodium hydroxide (pH » 8~LL) possessing a T/X'.'eped^.

- tect&lt;^eroxida^ by Slgna (St. Lcuis^Missairij Lot :15X/'ltor'2W5/'./. . Χ.?&lt; .'/X &lt;//./ך ! *.־ _v., - Hydrogen produced by Signa,. tot׳.'

NO. H-1009. χ. ' / /. ; , /.

X׳^’X. X׳־׳׳/ ///. X/ .X/./X.’־'X X־X ./'X//'/.^:1. '׳/fceinstrumentoutHized for ttew^ • , - Garana Ccunter/produced by Elscint, Israel (integrated Nuclear . 20 /־:Spectrometer, ׳model^INS-UN). '.

xX.? / ״'x . .. ’ ,. ׳ .״ יי’,.־' ’ ‘ ׳' * ^! X ־ / -Xtato/Samte 400 .(©, produi^ by^ ־ ; ׳ '׳. .X., . / >. :, - x- / ' .’.׳ .. . _ד , ־

- Cooled ultra Centrifuge, produced by Cryafuge, Heraeus,. West' Germany, .χ//,.’ X X'.'/;

.Λ t ., J .>.,.-.» .. t , י,.'. .* f . .: «. .' .;' , .׳ ' .-> ^;׳ י;׳*׳ v - Column SephadeX G-100 (9 x 550 mm).

‘.-׳;' ''-׳'יא'יl'//:'.'. '׳':.;.'J:. γ ׳ : ' .

A. tt-. Fraction Collector, produced ty Pharmacia, Frac-100 (Sweden).

Step, a ׳.:׳.A’יי;. ״׳ .< A AA A-י'׳ יא .,; :>/> .. ¹ ׳ ־׳ 'ATTA- τ A. 125.

The labellingwas done ty radioactive iodine ( X), at ,roan ten$»rature in a 2 mlplastic test tube, having at its bottom : aκ׳metal.׳rod operated ty a magnetic stirrer, which rotates’at' ',.A2 rotations per secoiids . The foUowing reagents were introduced יי' ז . 3 pg of placental protein in 10 pi 'of phosphate buffer saline '^,׳ ־ ' י' - י^,'. .;<-<> . .,י VY'A''‘ י ' . /י' T (pH » 7.2);

׳״ ־'׳ . .׳״. / ׳.' י, ׳ - ' ׳,׳׳' ’ ׳. .. ΐ '^,׳ י ׳'׳׳ י.''׳.

-/1 pg of lactcperoxidose in 6 pl of phosphate buffer saline;

’ ' .A' '. ' 125.λ . י'’.' ׳ A.־. A/ .γ.7.-;3^ίώ^- and/;' ' , / ־ <׳, v ' ’>. 1 . . . ' ',1׳ ,> — . ן ‘ . , ' _1י . . . ' nmole of hydrogen peroxide in 10 pl of phosphate buffer

A’ saline. /' ;A׳' A'. .. At.'C’ ׳

The reaction solution c»nsisted of 5 pg of PP-13 to which it was ^::. :?>.Α .^;A τΪ\.>.αΑ'''^/ .Α'-׳λΑ\־'. 125 י ''־A' A> י' . * added 1 pg of lactoperoxidaseand 325 pci of I. Due to the 'Ά ' *^J' ’ . י. ' ' ^% A ¹. '׳’־. r A ' ^Λ י' ז ' ‘ * ' . T . .יך .

relatlve high alkalinity prevailing thereto, an amount of 3 pl of phosphate buffer solution (0.4 M) vras added the pH reaching a

‘ '׳ ׳. . ״׳.י/.־-';.%.׳:־׳יי׳ ..,- .׳.׳ ;

Ara The iodination reaction started by the addition of hydrogen peroxideandcentinuedforabout 3minutes. The

Nal (2 g/1):. NaN (l g/l) and NaCl (8,5 g/1). After an additional . ץ׳’ י 3.'״. ., ־ ,.־.-’. ..a . a'. ־'^,־ αλΑ A‘.

/7 /7' ' 7<' ' ' י':- 7'147'^ר''^- י'' י''־ . ' 1. ־ ׳־ * . ¹ . י יי-.׳ . ,. ׳ ן* י _t > . . -7 ׳׳־ ־ י י. i . י . _t . ׳ far 1 rolmite; the raUture: G-100 column for extracting Mt the labelled !«rotein from the free Iodine

. . . ־ * , ׳*- ׳ !, * 7• ? ׳ ׳ ׳ . ׳ . 7 . י י ’ ׳ ./׳/*' ' and the other reagents, by a solutionconsistii^f of: phosphate bufferealine + 1% bovine serum albumen and 0.1% NaN . Prom the

׳' י'־' ' 3 ,. ? : :' / ' '7 ' . ''^;'7^V7^:;. ׳ 7' ’ ' ־. . י י ־ י “י . &lt; . /. , . .י.?. . . י'־^,, י,''' extent ©f iodination was determined by a ־the.׳ resulted' ifritetlons׳;'5•

Ganna Counter (Elscint) and the calculated yield was found to be

' ׳' ’ ' י:' / י' 7 7 'י/ /.׳: ' '7:' %7».64./-; י /

Inorder !» calculate the specific activity of the labelled antigen, samples oflO|ilwere taken and again counted by Packard 10 Gamma Caunter and found to be 86178 cpm/ng.

' Step b, י 7'/.';';. 7׳7־־' ';.?'7 ^י \ '

Staidaid solutiOTs of various c»hoentratlons in the range.' of 0.2 ng/ml to 64 ng/ml of antigen in a buffer (tris HCl, pH . 7.4, 0.02ή/i and NaN , 1 g/1) were prepared.

3.:. . י; 7• . י . :. . ־,,,. <.. _Λ ; 7 . . .;׳ , י י ׳ - -. ׳.'.>> ' ' ' .' ' ' ’;Λ י .'/' י

The labelled antigen consisted of a diluted solution of 1 ng/ml. . .. ' יי &lt;f . . , ; . /. י' ׳י. , , ' . ' . ... . ' I

The first antibody, was the antiserum of the placental antigen ; diluted in. a buffer (with the same composition as above) * :-7: 7.Step c. ^c lhe second antibody, was developed in the serum of a donkey. : The test #as!. parried 'out in a plastic test tube, the working scheme 20 tei1^ described in the follcwing Table 2. 77 ׳ / ' .

<img file="IL78237A_D0001.tif" />

TABLE 2, -ץ &lt; ^:. J' '' י /

The working scheme for the determination of pjgcentg] antigen in the human serum using radioimmunoassay.

<td> Kindof</td><td colspan="2"> Buffer A: Standards</td><td> Sample</td><td colspan="2"> Tracer First</td><td> Second</td>

<td> incubate ' ‘ ל</td><td colspan="2"> Tris-HCl,. ρΜ44(0.03β/1)♦</td><td></td><td colspan="3"> antigen antibody antibody . י _f v.;. ‘ .. - *z 4— --- . _״.</td>

<td> 1’ ί . ז _r . ‘ ..</td><td> NaN (1 g/1)</td><td></td><td></td><td></td><td></td><td></td>

<td> ״ . « ׳ י -.״¹. , . '</td><td> (ul) :/</td><td> (U1)</td><td> (U1)</td><td></td><td> (pl)</td><td> (ul)</td>

<td> '. Total . </td><td></td><td> -׳' ״י׳. 1 י ,ר , </td><td> ״τι¹¹¹¹¹¹ .׳״</td><td></td><td> יי-י^ז-״י</td><td></td>

<td> Radioactivity</td><td> 200</td><td> י י .&lt; ^י a. ’</td><td></td><td> 100</td><td> .» ' t ״ . .</td><td> **יי</td>

<td> Nonspecific</td><td> .י ^r'י . \ ז'־־ ל- י '</td><td> י ' &lt; י</td><td> .* .’</td><td> A-</td><td></td><td></td>

<td> binding ,</td><td> .ץ 200</td><td> '.־־**</td><td> «» .</td><td> 100 :</td><td> '**</td><td> 500</td>

<td> Zerobinding</td><td> ^00</td><td></td><td></td><td> 100</td><td> 100</td><td> 500’</td>

<td> .,StarKferds</td><td> '&lt;:/'!.;. . ,'4</td><td> י ׳ ₅ 10Q</td><td> י* .י! </td><td> 100</td><td> 100</td><td> 50()</td>

<td> ' ־'Srimplris ' *</td><td> .M</td><td> * .»</td><td> 100</td><td> 100</td><td> 100</td><td> 500</td>

V ‘ ־.'.. J. ' : ’ ־ . . ; ¢-- -, ' <. , . . >' t

The reactions between the labelled antigen with the sample of serum ''.;:or respective standards were carried out for about 18 hours are follcwed by the additiOT of the second antibody and agitated for .--.A. ' . * ’’ , : ' ¹ . .».’ ,. ‘ , *' . ' , ^,..י .

., about 10 minutes. The mixtures were centrifuged at room temperature' for. 30 minutes (at 1000 x C) arid the precipitates washed with 500 jul .,of phosphate buffer- 'sisline ^'agatn' C^trifUged^:''' Ihe radioactivity in the washed precipitate Was determined ' ' * ׳ s ' * *י ,״ . , * י t &lt; ' ' , ־ ' ' יי י , .

by Packard Gamma Qounter. The results obtained are gummariy^ in ^‘,.^.;/^.attached'/Flgw»: 1. It clearly appears from said Figure that an / antibody (A b) with a concentration of 1/50000, gave an adequate binding range for.an antigen concentration of between 0.4-32 ng/ml.

• '׳ .־ ' ' .. ’ ' - ׳ ' . ' I , / (־' ''.. ' . f . . '. .

In order to calculate the antlgen concentrations in the

-:.י samplestested, a. calibration curve was prepared (shown in :.

'W Ϊ ' 125 ־ '&lt;- ' '

Figure 2) correlating the radioactivity of I (expressed in counts per minute) versus the ΡΡ«.13 concentration (expressed in e . ng/rol). '7^: . --7 ג.'^,׳ י':.'.' ./'׳ ' λ '_'7’ό / ^^:'resMtscO&amp;teane^ ^four of population sables? adults males, adults non-pregnant feniales, aiult '(/, 7 7,..’ asymptomatic pregnant females and sdult syraptomatie pregnant .

^f '^: Y 10 females are iLLustrated in a graphlc manner in Figure 3. .