IE42036B1 - Pregnancy-specific antigens - Google Patents

Abstract

1492689 Anti - embryo serum UNIVERSITY OF MELBOURNE and VICTORIA STATE 17 Nov 1975 {18 Nov 1974 8 Aug 1975] 47248/75 Heading A5B [Also in Division G1] Anti-embryo serum for diagnosis of pregnancy is produced by immunising an animal with a homogenate of embryos or portions, extracts or purified fractions thereof or blood from a pregnant animal and purifying the resulting immune serum from irrelevant antibodies by absorption with tissues other than embryos, uterus, ovaries and blood from pregnant animals. Rabbits or calves are immunised with a homogenate of 12 - 14 day sheep embryos emulsified with an equal volume of Freund's adjuvant, repeatedly at weekly intervals, and blood is removed 6 days after each injection. Serum from the blood is purified from unwanted antibodies by absorption with homogenates of liver or kidney from non-pregnant sheep. The anti - embryo serum may be used for diagnosing pregnancy by mixing with whole blood, erythrocytes, serum or plasma from the animal being tested and examining the mixture by a method for detecting antigens, e.g. agglutination or agglutination - inhibition techniques, precipitation and immunofluorescence or radioimmunoassay tests.

Description

This invention relates to pregnancy-specific antigens and in particular to such antigens which can be applied.for the early diagnosis of pregnancy in mammals.

In humans, immunological tests for pregnancy 5 are known which tests give a positive reaction with anti-sera against human chorionic gonadotrophin (HCG) which HCG is present in blood or urine. lhe resulti of these tests are based on the agglutination by antibodies of sheep erythrocytes or latex particle in the presence of HCG.

It is an object of the invention to provide new anti-sera and a method of using these anti-sera for early diagnosis of pregnancy in mammals using ' · immunological techniques.

The invention includes an anti-embryo serum. ;;·;<..·· produced by the immunization of animal(s) with an homogenate of embryos, selected portions of embryosj extracts .of embryos, purified fractions of embryos or ,/ blood from pregnant animals and from which serum irrelevant antibodies have been removed by selective -.-. ,' .'it absorption with homogenates of tissues other than ·-. embryos, uteri, ovaries and blood from pregnant animals. ’ . .

The species-specific and other irrelevant r antibodies are preferably removed by successive absorption with homogenates of tissues other than embryos, uteri, ovaries and blood from pregnant animals. > ; - .

The invention also includes a.-method for -diagnosis of pregnancy in mammals comprising adding to blood from the animal an anti-embryo serum· produced . by the immunization of animal(s) with an homogenate of ' embryos, selected portions of embryos, extracts of . embryos, purified fractions of embryos or blood from ; ·' • - 2 42036 pregnant animals from which, serum irrelevant antibodies have been removed by selective absorption with homogenates of tissues other than embryos, uteri, ovaries and blood from pregnant animals, and examining the blood by a method of detecting antigens.

The preferred methods of detecting antigens are agglutination or agglutination inhibition techniques, precipitation reactions and radio-immunoassay techniques* The preferred, anti-sera are those raised against embryos of the species under test.

In this specification, the word blood is considered to include the fractions of blood, erythrocytes, serum and plasma and the words embryo can include all products of conception inoluding extra-embryonic membranes up to the time of birth.

When agglutination tests are being used, it is normal to test whole blood, erythrocytes, serum or plasma from the animal being tested, where agglutination inhibition tests are used, serum or plasma is used and the test is carried out on erythrocytes from another animal or latex particles onto which antigens or antibodies are absorbed, for precipitation and radio-immunoassay tests, either serum or plasma is used.

The invention also includes the testing to ascertain whether or not an animal is pregnant with multiple offspring by carrying out the process hereinabove described and ascertaining either by the occurrence of a stronger reaction such as stronger agglutination or by quantitation of the reaction after serial dilutions of the sera whether more antigen is produced than would be expected with - 2 43036 single offspring.

In many species of mammal s, -the functional life-span of the corpus luteum is prolonged during pregnancy. The stimulus for this prolongation must originate from the presence of an embryo in the uterus. However, the nature of the stimulus is hot known, 'further, if this stimulus could he easily t ( identified, it would provide a test for early pregnancy.

In the experiments hereinafter described, we refer to the u$e of the invention in association with sheep but it is believed that specific antiembryo sera oan be produced for other mammals in the same way in which the anti-serum for sheep was produoed.

In the sheep, an embryo must be present in the uterus on or before day 12-15 after mating if regression of the corpus, luteum is to be prevented. We hereinafter describe experiments to investigate the presence of antigens in the ewe which are specific to pregnancy and which might act as the stimulus involved in maintaining the corpus luteum.

Anti-embryo sera were produced by the immunization of rabbits with an homogenate of 12-14 day sheep embryos. The embryos were collected from pregnant ewes under anaesthesia induced by intravenous injection of sodium pentabarbitone and’maintained with an oxygen-fluothane mixture. The uterus and ovaries were exteriorized through a mid-ventral incision. Embryos were flushed out with a sterile 0.154M MaCI solution injected into the tip of one uterine horn and recovered through a glass'cannula inserted into the tip of the opposite uterine horn.

Normal embryos were either frozen at - 1O°O for use - 4 43036 in inoculations, or transferred to 10% phosphate-buffered formalin for subsequent histological examination.

Biopsies of endometrium, ovary, skeletal muscle, liver and kidney were taken from non-pregnant sheep on days 8-15 · after oestrus, and endometrium, ovary and skeletal muscle were taken from pregnant sheep on days 8-17 of pregnancy. Immediately after removal, these tissues were fixed in 10/ phosphate-buffered formalin.

Adult New Zealand rabbits were injected intramuscularly with homogenates of whole sheep embryos emulsified with an equal volume of Freund's complete adjuvant (C.S.L., Melbourne). The inoculation was repeated 4 weeks later. Subsequently, at weekly intervals, the rabbits received injections of embryo homogenate alone.

They were bled 6 days after each injection. Speciesspecific and other irrelevant antibodies were removed from anti-sera by successive absorptions with homogenates of liver and kidney from normal, non-pregnant sheep.

Sandwich fluorescent staining was used to localize antigens. Frozen sections (6 pm) of the fixed embryos were cut at-2O°G, treated with the absorbed or unabsorbed sera for 50 min. and washed in phosphatebuffered saline (0.145 NaCl, 0.01M phosphate at pH 7.1) for 20 min. with 2 changes of buffer. The sections were then stained with sheep anti-rabbit-globulin conjugated with fluorescein isothiocyanate (Wellcome Reagents ltd., England) for 50 min., washed again for 20 min. and subsequently mounted with phosphate-buffered glycerol for fluorescence microscopy. - 5 4S036 Tissues treated with, normal (preimmunization) rabbit serum and immune serum fully neutralized by absorption with embryo homogenates provided tests for specificity.

In Table 1, the tissues studied, the stage 5 of pregnancy of the animals from which these tissues were taken and the results of immuno-fluorescent staining are shown. From day-8 until day-17 of pregnancy, both the cytoplasm and the cell membranes of the embryonic tissue stained very intensely indicating the presence of a pregnancy-specific antigen. The fluorescence in othef tissues was: (a) intense on the endothelial lining of blood vessels and on the cells bordering the tissue spaces in the myometrium of the pregnant uterus, (,b) less intense in the cytoplasm of some luteal cells in the corpus luteum, and (e) intense on the membranes of erythrocytes present in all of these tissues on days 8-17 .of pregnancy.

In sections of skeletal muscle only the erythrocytes and occasionally the endothelial lining of blood vessels showed fluorescence. This staining of embryonic tissue, uteri, corpora lutea and erythrocytes was inhibited by the absorption of the antiserum with an homogenate of embryo or uterus, or erythrocytes from a day-14 pregnant sheep or by the neutralization of the antiserum with plasma from pregnant ewes. Repeated absorption with homogenates of liver or kidney or erythrocytes from non-pregnant sheep or the treatment of the antiserum with plasma from non-pregnant ewes did not inhibit fluorescence. This indicated that the antigen(s) were peculiar to pregnancy and were present in the uterus, corpus luteum and blood of ewes as early as day-8 of pregnancy and were present on the embryo. - 6 42036 The presence of antigen(s) on erythrocytes was shown by a simple haemagglutination test involving the agglutination of erythrocytes from ewes between day-8 and day-17 of pregnancy with the anti-pregnancy serum and will be described in detail later.

It was observed that the antigen(s) present in the plasma of ewes at day-14 of pregnancy would bind to the erythrocytes of castrated male sheep (wethers) and these coated erythrocytes agglutinated in the presence of the anti-serum. A direct haemagglutination test to monitor antigen titre was thus developed and will be described in detail later. These antisera were of the non-precipitating type, and no reaction was observed in gel diffusion with either homogenates or plasma from pregnant ewes.

Subsequent antisera, producing by immunizing 8 rabbits and 2 calves with homogenates of sheep embryos, after a series of absorptions with tissues from nonpregnant ewes, were'found by immunofluorescence to react in a similar way to the initial antisera and localize in the uterus and corpus luteum and embryo. However, the antisera produced in the calves and 5 of the rabbits were precipitating and after absorption with tissues from non-pregnant sheep were observed to precipitate in agar gels with an homogenate of 14-day sheep embryo.

As yet, these antisera have not been assessed in an haemagglutination system.

Preliminary characterization of the antigen(s) has been oarried out using uterine vein and peripheral plasma, and in some cases erythrocytes, uterus and corpus - 7 42036 luteum from ewes at day-14 of pregnancy. Immunofluorescence was used to monitor the presence of antigen(s) in uterus and corpus luteum, while the simple haemagglutination test and direct haemagglutination test were used to monitor the presence of antigen(s) on the erythrocytes and in the plasma respectively.

The antigen(s) in plasma are stable at 37°0 for at least 4 hours, at 4 °C for at least 1 week and at -15°C for at least 12 weeks. The activity was slightly reduced after heating at 56°C for 30 min. and'was destroyed after heating at SO°C for 30 min. Following dialysis (MW cut off 6000-8000) against phosphate buffered saline (pH 7.1) overnight at 4°G,. the titre -of antigen in dialysed plasma remained unchanged. Filtration of plasma from ewes at day-14 of pregnancy on Sephadex G-25 (Registered Trade Mark-fractionation range 100-5000MW) showed that the anlligen(a) were present in the main protein fraction collected in the exclusion volume of the column. This indicated that the antigen(s) had a molecular weight of greater than 5000. Further chromatography on Sephadex G- - 100 (fractionationrange 5000-100,000 MW) showed that the antigen(s) were separated by the column into a band which emerged after the exclusion volume of the column indicating a molecular weight of between 5000- and 100,000.

Ammonium sulphate precipitation revealed that 45% saturation was the lowest effective concentration which removed the antigen(s) from solution.

Electrophoresis on cellulose acetate membranes at pH 8.5 indicated that the antigen(a) had an electrophoretic mobility in theα-globulin region. - 8 42036 Treatment of pregnancy plasma with M-trichloroacetic acid completely removed the antigen(s) from eolation, indicating a protein component is necessary in the antigen(s) for reaction with the anti-serum. No ahtigen(s) were detected in plasma which had been treated with sodium metaperiodate at concentrations of 0.05M to 0.20M. Also erythrocytes, after treatment with periodate in I concentrations between 0.00125M and 0.02M, no longer agglutinated with the antiserum. It therefore appeared that the antigen(s) contained a carbohydrate moiety which was essential for the reaction of the antigen(s) with the specific anti-pregnancy serum.

It was therefore decided to subject the plasma, erythrocytes and uterus and corpus luteum of ewes at day-14 of pregnancy-to various enzyme treatments. following treatment of plasma, erythrocytes and uterus and corpus luteum with papain or trypsin, there was no reaction of any of these tissues with the antiserum indicating that the antigen(s) contained a protein component. Treatment with neuraminidase also prevented any reaction with the antiserum, indicating that the antigen(s) contained a carbohydrate with sialic acid moieties. In contrast, after treatment with α-amylase, the antigen(s) were still detectable with the antiserum, indicating that 2b the antigen(s) dia not contain an «-1-4 linked glucose polymer.

These preliminary studies of the pregnancyspecific antigen(s) which were detected immunologically in the tissues cf pregnant ewes, indicated that they were of a molecular weight between 5000 and 100,000 and that they have a protein and carbohydrate component. It was shown further that the antigen(s) can be extracted from the 14-day embryo by soaking the embryo in water. After soaking overnight in distilled water, the cells of the embryo were swollen and - 9 43036 markedly vacuolated but not lysed and the antiserum no longer localized on the cells. After soaking overnight in phosphate-buffered saline, the antiserum was observed to localize on the cells of the embryo indicating that the ahtigeh(s) were not removed by the buffered saline and the microscopic appearance was similar to that of an untreated embryo.

The time-course of appearance of the antigen(s) on the erythrocytes of sheep was studied using the initial antisera and the simple haemagglutinated tests and blood collected ‘from sheep at various stages of their aestrous cycles and during pregnancy(Table 2) A 5% (v/v) suspension of erythrocytes in phosphate-buffered saline (O.145M NaCl, 0.01M phosphate at pH 7»1) was I prepared and pipetted dropwise into blood agglutination , I trays. One drop of a 1 in 16 dilution of: (a) the santisera which were made specific for the pregnancy antigen by repeated absorption with tissues other thai| conceptus, (b) the antisera absorbed with an homogenate of 12-day conceptus to remove pregnancy-specific antibodies, or (c) preimmunization serum from the rabbit, was added, mixed and allowed to stand for 30 min.

Erythrocytes taken from ewes between days and 50 of pregnancy showed agglutination with antiserum absorbed with tissues other than conceptus thereby indicating the presence of the pregnancy-specific antigen in blood (Table 2),. No agglutination of erythrocytes occurred with preimmunization serum or serum absorbed with embryonic tissues. There was no agglutination with erythrocytes from 2 rams, 4 wethers or from 12 non-pregnant ewes at any of the stages of the - 10 42 03« oestrous cycle examined. Also, erythrocytes from pigs, cows and horses in early pregnancy did not agglutinate with the anti-serum raised against sheep embryos, ilhis suggested that the antibody was specific to pregnant ewes. t, The detection of a pregnancy-specific antigen offers a simple test for the diagnosis of pregnancy at day-6 in the ewe.

In a further experiment to determine the accuracy of this simple haemagglutination test, 550 Ί0 ewes which had been mated up to 55 days earlier were tested and pregnancy confirmed at slaughter. An unmated control flock was also tested at monthly intervals for 4 months over the same period. In the mated ewes, 64/ were correctly diagnosed positive / 23/750} and negative (8^/550 ) over the period of observation.

There were °^/33O (19/) false positive tests in the mated flock, of which 55/ were attributed to ewes in which embryo death and resorption was considered to have occurred subsequent to testing, lhe remaining 45/ of false positives oq may be accounted for by operator error in embryo recovery from slaughter material and in reading the test. There were ^/330 (17/) false negative tests in the mated flock which, when examined relative to the stage of pregnancy it become apparent that the incidence of false negatives increased in the 41-50 day period, suggesting that the presence of excess unbound antigen in plasma may have interfered with the haemagglutination. The accuracy of the haemagglutination test at different stages of pregnancy was as follows: 6-12 days of gestation-61/ correct positive; -50 days - 72/1 21-50 days - 78/: 31-40 days - 85/ 4303θ 41-50 days - 44%. In the unmated control flock there was a low incidence (6%) of false positive tests. .

The inability of the simple haemagglutination test to distinguish'between high and low titres of antigen indicated the necessity of using an alternative haemagglutination procedure to improve the accuracy of diagnosis. ,, The alternative procedure was the direct haemagglutination test which was previously shown to -jQ detect antigen(s) in the plasma of ewes at day-14 of pregnancy. Plasma samples from 81 ewes were collected over .62 days after mating. Plasma (50 ul) was incubated at 57°C with 50 Ul pf a dilution (e.g. ^/16) of rabbit 1 I anti-sheep embryo serum. After 1 hour, 50 μ 1 of a 2.5% I (v/v) suspension of erythrocytes from castrated male sheep (wethers) was'added and the mixture incubated at 4°C for 20 min. when the erythrocytes were assessed for agglutination. This binding haemagglutination test i ζ-λ diagnosed 89% of animals correctly (ou/81 positive and 12 /81 negative) at 62 days of pregnancy, there being 5 false positive test and 4 false negative tests.

Of the ewes confirmed pregnant at slaughter 62 days after mating, only 10% had been diagnosed positive between 0-6 days, 29% between 7-12 days, 47% between 14-20 days, 75% between 21-27 days, 76% between 28-24 days, 95% between 25-41 days, 71% between 42-48 days, 100% between 49-55 days and 85% between 56-62 days. The low accuracy before 20 days of pregnancy probably reflects insufficient titre of antigen to attach to the erythrocytes for agglutination to occur. On the other hand, this binding haemagglutination test is much more accurate than the simple test after 40 days of gestation. - 12 42936 Four of the 5 ewes with false positive tests at 62 days of pregnancy were thought to have lost their embryos but still had residual quantities of antigen in their blood, In view of the complications of embryo resorption which occurs mainly before 25 days of pregnancy the binding haemagglutination test is more suited to diagnosing pregnancy between days 30-65.

Already, antisera which have been shown by immunofluorescence and gel diffusion to. react with antigen(s) in now embryos have been produced by immunizing rabbits with a homogenate of 30-day to 40-day cow embryo. Following absorption with homogenates of liver and kidney from non-pregnant cows, the antisera still reacted with an homogenate of cow embryo (30-day to 40-day) by precipitation in agar gels.

In another experiment an anti-serum specific to pregnancy in horses has been raised by immunizing rabbits with blood from a pregnant mare. Following absorption procedures similar to those previously described the anti-serum was shown to be active.

It will be appreciated that the rapid identification of pregnancy in animals can be of great value, the value depending upon which animals are tested. For example, if the pregnant ewes from a flock of sheep can be readily identified, then it is possible to immediately remove these from the flock thus increasing the reproductive efficiency of the flock as a whole. Further, if ewes carrying more than one foetus can be identified, it is possible to separate these ewes so that they can be managed separately from the remainder of the flock again resulting in an improvement in reproductive efficiency. - 13 -. 43036 It will be seen that the antisera which are specific to pregnancy in cows can be of great value as in many cases cows are artificially rather than naturally inseminated and the dse of such an anti-serum can give an early indication aB to whether.or not fertilization has been effected and if not the animal can be treated so that artificial, insemination can be again attempted l within three weeks, further, the early identification of pregnant cows enable the reproductive efficiency 10 of the herd to be increased by special management.

Also, with cattle, the knowledge that a particular cow is pregnant with twins enables special management to ensure the health of both twins and cow.

When the invention is applied to horses, it has the first advantage of the use of the invention with respect to cattle, that is if pregnancy is not found, insemination can be repeated. In horses, breeding twins is not considered desirable and if there is an indication the mare is pregnant with twins, then a decision can be 2° ’made as to whether or not to abort.

The same advantages apply for pigs, dogs and goats as for sheep and cattle. for humans, the test appears to have all the advantages of any previous pregnancy test. It may also have advantages over other immunological tests which rely on a measurement of HCG which can give positive results for a non-pregnant condition, e.g. when a hyatidiform mole, chorio adenoma or chorio carcinoma is present. fci, ω rt PM o rt Ώ rt a) ω ra ra ra o •H rJ +5 rt gO •ri 0) hfl Pi Ρί ω •Η (I) q .ti •r| ra tri I Ρ ·Η ra p P S CD rC| ϋ -p ra ·Η O rd d ra H +3 -P >> q -p tri ra 6b rt φ CD Pl •p Pi o P rt i> •rt iJ s§ r-1 Q) iM iers in. brackets refer to the number of animals examined on the day indicated ι Day of oestrus.

(U £ TABES 2 agglutination with anti-sheep embryo sera of erythrocytes from pregnant and. non-pregnant ewes, rams and wethers.

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Claims (15)

1. WHAT WJi CIAIM IS:1. An anti-embryo serum produced by the immunization of animal(s) with a homogenate of embryos, selected portions of embryos, extract of embryos, purified fractions of embryos or blood from pregnant animals from which serum, irrelevant antibodies have been removed by selective absorption with homogenates of tissues other than embryos, uteri, ovaries and blood from pregnant animals,

2. An anti-embryo serum as claimed in claim 1 wherein the homogenised tissue used for absorption has been derived from non-pregnant animals.

3. An anti-embryo serum as claimed in either claim 1 or claim 2 for use against a particular species, the serum having been raised against embryos, selected portions of embryos, extracts of embryos, purified fractions of embryos or blood from pregnant animals of the particular species.

4. An anti-embryo serum as claimed in any one of claims 1 to 3 wherein the selective absorption comprises successive absorptions for different irrelevant antibodies.

5. An anti-embryo serum as claimed in any preceding claim, wherein the homogenates of embryos, selected portions of embryos, extracts of embryos, purified fractions of embryos or blood from pregnant animals have been emulsified with an adjuvant.

6. An anti-embryo serum as claimed in claim 5 wherein the adjuvant is Freund’s adjuvant.

7. An anti-embryo serum as claimed in claim 4 wherein the successive absorptions are with homogenates of liver or kidney of the particular species or erythrocytes of non-pregnant animals of the particular species. - 17 42036

8. An anti-embryo serum as ! claimed in claim 7 wherein there are repeated absorptions with any of the homogenates.

9. An anti-embryo serum as claimed in any 5 preceding claim wherein the homogenate has been injected into animals which are subsequently bled, the serum being separated. from the remainder of the blood prior to the removal of the irrelevant antibodies.

10. A method for diagnosis of pregnancy in 10 mammals comprising adding to blood from the animal an anti-embryo serum'according to olaim 1 and examining the blood by methods for detecting antigens.

11. A method for diagnosis of pregnancy in mammals as claimed in claim 10 wherein the method 15 for detecting antigens comprises agglutination techniques, agglutination inhibition techniques, precipitation reactions and raidoimmunoassay techniques,

12. A method of diagnosis of pregnancy in mammals, as claimed in claims 10 and 11, wherein the 2o quantitation degree of the antigen indicates no pregnancy, a single pregnancy or a multiple pregnancy.

13. A method of diagnosis of pregnancy in mammals as claimed in any one of olaime 10 to 13 wherein the serum is raised against embryos, selected 25 portions of embryos, extracts of embryos, purified fractions of embryos or blood from a pregnant animal of the species being diagnosed.

14. An anti-embryo serum according to claim 1 and substantially as hereinbefore described. - 18 42036

15. Λ method of diagnosis of pregnancy in mammals according to claim 10 and substantially as hereinbefore described.