AU599021B2 - Method for detecting early pregnancy factor (epf) in mammals,purifying epf and method for producing a monoclonal antibody - Google Patents

Description

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AU-Al 7 PCT WORLD INTELLECTU

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INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 C07K 15/06, 15/12, 3/20 C12N 15/00, GOtIS 33/577 N' (11) International Publication Number: A1 (43) International Publication Date: 25 S W 0 86/05498 eptember 1986 (25.09.86) (21) International Application. fiber: PCT/AU86/00060 (22) International Filin.-e': 12 March 1986 (12.03.86) (31) Priority Application Numbers: PG 9664 PG 9750 PH 2402 (32) Priority Dates: (33) Priority Country: 12 March 1985 (12.03.85) March 1985 (15.03.85) 12 September 1985 (12.09.85) (74) Agent: GRANT ADAMS COMPANY; 333 Adelaide Street, Box 1413), Brisbane, QLD 4000

(AU).

(81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (Eurooean patent), FR (European patent), GB, GB (European patent), IT (European patent), JP, LU (European patent), NL (European patent), SE (European patent), US.

Published With international search report.. With amended claims.' A.0.J.p. 6 NOV 1986 i= a AUSTRALIAN 130CT1986 PATENT OFFICE ay. *F (71) Applicant (for all designated States except US): UNI- VERSITY OF QUEENSLAND [AU/AU]; St. Lucia, QLD 4067 (AU).

(72) Inventors; and Inventors/Applicants (for US onlv) MORTON, Halle [AU/AU]; 75 Orion Street, Cooparoo, QLD 4151 CAVANAGH, Alice, Christina [AU/AU]; 29 Anderson Avenue, Ashgrove, QLD 4060 (AU).

ROLFE, Barbara, Ellen [AU/AU]; 7 Birdwood Street, Ipswich, QLD 4350 (54)Title: METHOD FOR DETECTING EARLY PREGNANCY FACTOR (EPF) IN MAMMALS, EPF AND METHOD FOR PRODUCING A MONOCLONAL ANTIBODY

PURIFYING

(57) Abstract Cells which produce EPF are grown in a culture medium to produce a supernatant medium containing the EPF. To purify the EPF, the EPF is absorbed by a selective absorbent in a column, dialysed against a buffer solution, concentrated and gel-filtered. Selected fractions of the filtrate undergo reversed phase high performance liquid chromotography and the purified EPF is eluted from the chromotography column. Monoclonal antibodies to EPF can be produced to detect the presence of EPF in serum and provide a means for detecting pregnancy in female mammals.

i WO 86/05498 PCT/AU86/00060 -1- Title: "METHOD FOR DETECTING EARLY PREGNANCY FACTOR (EPF) IN MAMMALS, PURIFYING EPF AND METHOD FOR PRODUCING A MONOCLONAL ANTIBODY" BACKGROUND OF THE INVENTION Field of the Invention This invention relates to a method for detecting early pregnancy factor (EPF) in mammals, purifying EPF and a method for producing a monoclonal antibody therefor.

Prior Art Most home pregnancy kits can only detect and indicate pregnancy approximately 3-4 weeks after fertilization.

Pregnancy involves two early important milestones fertilization of the ovum and implantation of the fertilized ovum in the uterus approximately eight-ten days after fertilization.

It would greatly assist research if the precise times that both milestones occurred could be detected. It would also be an advantage if a woman was aware she was pregnant immediately after fertilization so that she could avoid e.g. smoking, alcohol, surgery and x-rays or radiation treatment.

It has been established that EPF is produced within 24 hours of fertilization but the problem has been to isolate the EPF protein, purify it and produce an antibody which can be used to detect the presence of EPF e.g. in serum or urine as a test for pregnancy.

BRIEF SUMMARY OF THE INVENTION With the above matter in mind, preferred objects of the present invention are to achieve methods to overcome each of the problems hereinbefore described.

In one aspect the present invention resides WO 86/05498 PCT/AU86/0006 0 -2in a method for producing EPF from any mammalian cell source including the steps of: growing a selected cell which produces EPF in a culture medium to produce a supernatant medium containing EPF and other products; and harvesting the supernatant medium to obtain the

EPF.

In a second aspect the present invention resides in a method for purifying EPF including the steps of: passing a supernatant medium containing EPF through a column containing a selective absorbent for the EPF immuno absorption of the EPF); eluting the EPF from the selective absorbent to produce a first eluate; effecting reversed phase high performance liquid chromotography (HPLC) on the first eluate in a column; and eluting the bound EPF from the column to collect the purified EPF.

Preferably the first eluate is dialysed against a buffer solution to remove any small molecular weight products; the dialysis product is concentrated; gel filtration is effected on the concentrate; and selected fractions of the filtrate are collected and the reversed phase high performance chromotography is effected on the collected fractions.

In a third aspect the present invention resides in a method for producing monoclonal antibodies to EPF including the steps of: immunizing an animal with purified EPF; removing the spleen of the animal and fusing the spleen cells with selected cells; growing the fused cells in a culture medium; m WO 86/05498 PCT/AU86/00060 -3selecting the hybrid (fused) cells from the non-fused cells; and cloning out the hybrid cells producing the EPF antibody by limiting dilution methods.

The hybrid cells may be grown in a culture medium in vitro) to produce high concentrations of monoclonal antibody to EPF, or in BALB/C mice in vivo) and harvesting from these mice serum and/or ascites which will contain high concentrations of monoclonal antibody to EPF.

In a fourth aspect the present invention resides in a method for pregnancy diagnosis in a female mammal including the steps of: mixing EPF antibodies with serum or urine believed to contain EPF; and monitoring any reaction due to the presence of EPF in the serum or urine.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT To enable the invention to be fully understood, a preferred example will be described with reference to human EPF.

Human EPF was produced by continuously growing Choriocarcinoma cells (sold under the trade mark "Be Wo" by the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. and deposited under ATCC Deposit No. CCL98), human myeloma cells (ATCC Deposit No. CCL155) or human lymphoblastic leukemia cells (ATCC Deposit No. CRL1582) in culture medium comprising "DMEM" ("Dulbecco's Modification of Eagle's Medium") (sold by Flow Laboratories Inc., 7655 Old Springhouse Road, McLean, VA 22102, U.S.A.

and subsidiary companies in,inter alia Australia, Canada, Japan, the Uni.ted Kingdom and Sweden), and foetal calf serum (sold by Commonwealth Serum Laboratories, Melbourne, Australia) and harvesting the

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WO 86/05498 PCT/AU86/00060 -4supernatant medium. This supernatant medium contains human EPF and other products.

An immuno-absorption column is prepared using goat/anti-mouse EPF (or any other suitable EPF e.g.

using rabbits or donkeys as the host for mouse, human or rat EPF).

To prepare the column, 800mg. of anti-EPF IgG (immunoglobulin) is absorbed with human serum and with foetal calf serum bound to cyanogen bromide activated Sepharose 4B (sold under the trade name "CNBr-activated Sepharose 4B" by Pharmacia Biotechnology Products, Sweden see page 91 of their "Catalogue 86") and the absorbed IgG is bound to 30mL of "Affigel- (trade name of Bio-Rad, 2200 Wright Avenue, Richmond CA 94804, U.S.A. and subsidi-ry companies in, inter alia, Australia, Canada, Japan, the United Kingdom and Switzerland. "Affigel-10" is detailed at pages 46-51 of the Bio-Rad Catalogue No. K1985). A pre-column is placed in series with the absorption column having 4g. of IgG from normal male goat serum bound to 100mL. of The supernatant medium, containing the EPF, is pumped through the column and the human EPF will bind to the anti-mouse EPF in the absorption column, the latter acting as a selective absorber of the human EPF.

The human EPF is eluted with 1M acetic acid/ 0.9% NaC1/10% dioxane and the eluate is dialysed and the buffer is exchanged for 1M acetic acid adjusted to by ammonium hydroxide to remove small molecular weight products. (All percentages are expressed as or The product is concentrated to a 3mL. volume and undergoes gel filtration in a column of Sephacryl S-200 (supplied by Pharmacia Biotechnology Products, Sweden see page 84 of their"Catalogue 8 6")which has rP WO 86/05498 PCT/AU86/00060 been equilibrated with 1M acetic acid adjusted to with ammonium hydroxide.

This sample is filtered on the basis of molecular size and, on a 16mm. x 900mm. column, collecting 2mL. fractions, fractions 60-80 120- 160mL. flow) contain the EPF. These are pooled and TFA (trifluroacetic acid) added to a final concentration of 0.1%.

The mixture is applied to a Beckman RPSC ultrapore reversed phase HPLC column 4.6mm. x which has been equilibrated with 0.1% TFA.

The bound EPF is eluted with a 2 minute linear gradient to 25% isopropanol followed by a 30 minute /inear gradient to 30% isopropanol both containing 0.1% TFA at a flow rate of ImL./minute.

With the inclusion of a 2mL sample loop in the solvent path of the columns, the fractions eluted with retention times between 9.8 11.3 minutes contain the purified EPF. These fractions are pooled and stored.

With some immunoabsorbents, it is possible to omit the dialysis and gel filtration steps and apply the eluate directly to the reversed phase HPLC column (which has been equilibrated with 0.1% TFA), after addition of TFA to a final concentration of 0.1%.

To produce the monolconal antibodies to the human EPF, BALB/c mice bred from BALB/cJ strain mice from The Jackson Laboratory, Bar Harbor, Maine, 04609, were immunized with the purified EPF e.g. with 4 to 5 injections at monthly intervals.

The spleens were removed and the spleen cells were fused with mouse myeloma cells catalogue Nos. X63-Ag8-653 or Sp2/0-Ag-14 from Flow Laboratories Australasia Pty. Ltd., 140 Wicks Road, North Ryde, Sydney, N.S.W. 2113, Australia).

The cells are grown in "DMEM" medium with WO 86/05498 PCT/AU86/00060 -6- 2mM fresh L-glutamine,20% foetal calf serum plus antibiotics and fungicides, and the hybrid cells selected by addition of HAT medium (containing M hypoxanthine/4 x 10~ 7 M aminopterin/1 6 x 10-5M Thymidine).

The hybrid cells are cloned out by limiting dilution techniques and are tested to establish which clones produce an antibody to EPF. As hereinbefore described, the hybrid cells may be grown in a culture medium in vitro) to produce high concentrations of monoclonal antibody to EPF, or in BALB/c mice in vivo) and harvesting from these mice serum and/or ascites which will contain high concentrations of monoclonal antibody to EPF.

Those cells are recloned until a cell producing a monoclonal antibody is achieved. Samples of clones in intermediate or final stages are stored in liquid

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2 The resultant product can be used to detect EPF in human serum or urine for pregnancy diagnosis.

A number of alternative methods for diagnosing pregnancy through the detection of EPF in serum will now be described.

In a liquid phase method, EPF (including some labelled with [1251] is mixed with anti-EPF antibody and the mixture is allowed to react to produce a complex. The complex is precipitated by adding a precipitating antibody or polyethylene glycol.

The presence of any Iodine 125 in the precipitate .1 is maonitored using a ,-counter.

A negative sample no EPF in the serum) plus the labelled EPF and the antibody will result in a high (count while a positive sample (containing EPF) plus the labelled EPF and the antibody will result in a low count. This is due to competitive binding f" A'0o 86/05498 PCT/AU86/00060 -7between the EPF and labelled EPF with the antibody, as the EPF will prevent the labelled EPF from binding.

In a solid phase method, two different antibodies to EPF may be used, the antibodies binding to different sites on the EPF molecule. One antibody is placed in a plastic tube or on polystyrene beads or sticks and allowed to bind. The serum is added to the tube or placed in contact with the beads or sticks and EPF therein is allowed to bind with the first antibody. A second antibody, labelled with Iodine 125, is then allowed to bind with EPF. The bound Iodine 125 is counted with a Y -counter and a high count indicates the presence of EPF.

This method would be particularly suitable for a home pregnancy testing kit where the first antibody is bound on a testing stick which is dipped into the female's urine specimen and then into a first container supplied with the kit containing the second antibody labelled with an enzyme which undergoes a colour change when the stick dipped into a second container supplied with the kit containing a suitable substrate.

It will be readily apparent to the skilled addressee that the embodiments described above are specific and that a range of chemical proportions and times may be used. For example, the gel filtration may be carried out using "Sephadex G-100" (see page 80 of the Pharmacia Biotechnology Products "Catalogue 86") equilibrated with 1M acetic acid While human EPF has been described, the methods are suitable for the EPF of all mammalian animals. In particular the method for detecting pregnancy can be extremely important in the horse and cattle industries and in the preservation of endangered species. For example, the giant panda gives no indication of pregnancy but pregnancy could be determined, withr i i

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WO 86/05498 PCT/AU86/00060 -8out handling the female, by collecting urine e.g.

from the cage floor, and assaying with the particular suitable monoclonal antibody.

Various changes and modifications may be made to the embodiments described without departing from the scope of the present invention defined in the appended claims.

Claims (25)

1. A method for producing early pregnancy factor (EPF) from a mammalian cell line comprising the steps of: growing a mammalian cell line in a culture medium to produce a supernatant medium containing EPF and other products; and harvesting the supernatant to obtain the EPF.

2. A method as defined in clrim 1 characterised in that: to produce human EPF, the selected cells are choriocarcinoma cells, human myeloma cells, human lymphoblastic leukemia cells or a combination of two or more of these.

3. A method as defined in claim 1 or claim 2 characterised in that: to produce human EPF, the culture medium 20 comprises "Dulbecco's Modification of Eagle's Medium" and foetal calf serum.

4. A method for purifying EPF wherein the step of harvesting the supernatant medium to obtain the EPF as defined in any one of claims 1 to 3 is *ao characterised by the steps of: passing the supernatant medium through a column containing a selectivei absorbent for the EPF; 30 eluting the EPF from the selected absorbent produce a first eluate. effecting re~e~rd jhas. high esaormanc liquid chromotogr,,p t istst eluate in a column; and eluting the bf column to collect the pi A method as defined in claim 4 characterised in that: the first eluate is dialysed against a buffer solution to remove any small molecular weight products; the dialysis product is concentrated; gel filtration is effected on the concentrate; and selected fractions of the filtrate are collected and the reversed phase high performance chromotography is effected on the collected fractions.

6. A method as defined in claim 5 characterised in that: the gel infiltration is effected in a column containing a gel filtrate equilibrated with IM acetic acid adjusted to pH

7. A method as defined in any one of claims 4 to 6 characterised in that: for producing human EPF, goat/anti-mouse EPF, goat/anti-human EPF, goat/anti-rat EPF, rabbit/anti-human EPF, rabbit/anti-rat EPF, donkey/anti-mouse EPF, donkey/anti-human EPF, donkey/anti-rat EPF, mouse/anti-human FPF, or a combination of two or more of these is used as the selective absorbent for the EPF. Oe 6 e 20 i 0 e Q 6 6*6 6 @660 6*66 6 66 6 6060 6* 6 6*6 0 30 8. A method as defined in any one of claims 4 to 7 characterised in that: for producing human EPF, the human EPF is eluted with 1M acetic acid/0.9% dioxane and the buffer solution is exchanged for 1M acetic acid adjusted to pH 3.0 by ammonium hydroxide. Ll

9. A method as defined in any one of claims 4 to 8 characterised in that: the selected filtration fractions are pooled and trifluroacetic acid is added to a concentration of and applied to a reversed phase HPLC column which has been equilibrated with 0.1% trifluroacetic acid. A method as defined in any one of claims 4 to 9 characterised in that: the bound EPF is eluted with a 2 minute linear gradient to 25% isopropanol followod by a minute linear gradient to 30% isopropanol both containing 0.1% trifluroacetic acid at a flow rate of ImL per minute.

11. A method as defined in claim 9 or claim characterised in that: with the inclusion of a 2mL sample loop in the 20 solvent path of the column, the fractions eluted with retention times between 9.8 11.3 minutes contain the purified EPF and these fractions are pooled and stored.

12. A method as defined in any one of claims 4 to 11 0 g characterised in that: otrifluroacetic acid to a final concentration of 0.1% is added to the first eluate beifore the chromotography step; and the chromotography step is effected in a reversed phase HPLC column which has been equilibrated with 0.1% trifluroacetic acid. C

13. A method for producing monoclonal antibodies to EPF characterised by the steps of: immunizing an animal with purified EPF obtained by the method as defined in any one of claims 4 to 12; removing the spleen of the animal and fusing the spleen cells with selected cells; growing the fused cells in a culture medium; selecting the fused cells from non-fused cells in a medium; and cloning out the hybrid cells producing the EPF antibody by limiting dilution methods.

14. A method as defined in claim 13 characterised in that: the hybrid cells are grown in vitro in a culture medium to produce a high concentration of monoclonal antibody to EPF. 20 15. A method as defined in claim 13 characterised in that: the hybrid cells ari grown in vivo in mice and *e are harvested from the mice serum and/or ascites which contains high concentration of monoclonal antibody to EPF.

16. A method as defined in claim 15 characterised in that: the mice are immunized with 4 to 5 injections 30 at monthly intervals.

17. A method as defined in any one of claims 13 to 16 characterised in that: the selected cells are mouse myeloma cells; the culture medium comprises "Dulbecco's Modification to Eagle's Medium", 2mM fresh L- I 000 9. 20 13 glutamine, 20% fresh foetal calf serum, antibiotics and fungicides; and the hybrid cells are selected in a HAT medium to kill any background cells.

18. A method as defined in any one of claims 13 to 17 characterised in that: the cloned hybrid cells are tested to establish which clones produce, an antibody to EPF; and these cells are recloned until a cell producing a monoclonal antibody is achieved.

19. A method for diagnosing pregnancy in a female mammal characterised by the steps of: mixing antibodies produced by the method as defined in any one of claims 13 to 18 with serum or urine of the female mammal believed to contain EPF; and monitoring any reaction due to the presence of EPF in the serum or urine, the presence of EPF indicating pregnancy in the female mammal. A method as defined in claim 19 characterised in that: the EPF in the serum or urine is used with an anti-EPF antibody with the addition of a trace amount of purified EPF labelled with Iodine 125; the mixture is allowed to react to form a complex; the complex is precipitated out; and monitoring the presence of any Iodine 125 in the precipitate using a f- counter, a low count indicating the presence of EPF in the serum or urine. 666* 6eO Se 0 609* 6 OSS6 6 14

21. A method as defined in claim 19 characterised in that: a first anti-EPF antibody is bound to a plastic tube, stick or beads; the serum or urine is placed in contact with the first antibody to enable any EPF in the serum or urine to bind with the antibody; a second different anti-EPF antibody, labelled with Iodine 125, is placed in contact with the EPF to bind therewith; and the presence of bound Iodine 125 is monitored with a X-counter, a high count indicating the presence of EPF.

22. A method as defined in claim 19 characterised in that: 20 o S2 a first anti-EPF antibody is bound to a plastic stick; the stick is dipped in the serum or urine to enable any EPF therein to bind with the first antibody; and the stick is dipped into a second different anti-EPF antibody labelled with an enzyme and then into a substrate which undergoes a colour change if EPF is present in the serum or urine.

23. Purified EPF obtained by the method of any one of claims 4 to 12.

24. Monoclonal antibodies to EPF obtained by the method of any one of claims 13 to 18. s e 4666 0 50 0S S 1 PI- I/ 20 25 A pregnancy testing kit for female mammals comprising: a plastic tube container, stick or beads to which is bonded a first anti-EPF antibody; a first container containing a second different anti-EPF antibody labelled with an enzyme; and a second container containing a substrate; wherein: after serum or urine from the female mammal is placed in the tube, or the stick or beads are dipped in the serum or urine from the female mammal, to enable any EPF therein to bind with the first anti-EPF antibody, and the second antibody and then the substrate are placed in the tube, or the stick or glass beads are dipped in the second anti-EPF antibody and then into the substrate, a change of colour of the substrate indicates the presence of EPF in the serum or urine and that the female mammal is pregnant; and wherein; said first and second anti-EPF antibodies have been prepared by the method as defined in any one of claims 13 to 18.

26. A method of pregnancy diagnosis, said method comprising; mixing an anti-EPF antibody with serum or urine from the mammal believed to be pregnant; and monitoring the reaction to determine if EPF is present in the serum or urine. o* 9 .0. 999 9

27. A method as defined in claim 26 wherein said anti- EPF antibody is a polyclonal antibody. 16

28. A method as defined in claim 26 wherein said anti- EPF antibody is a monoclonal antibody.

29. A method as defined in any one of claims 26 to 28 wherein said reaction is monitored by using either a second polyclonal or monoclonal antibody. Purified EPF substantially as hereindescribed with reference to the example.

31. Monoclonal antibodies to EPF substantially as hereindescribed with reference to the example.

32. A method of purifying EPF substantially as hereindescribed with reference to the example. DATED this fifth day of April 1990. C. S. 00 SO SS 0 S S S. S S S UNIVERSITY OF QUEENSLAND by its Patent Attorneys GRANT ADAMS COMPANY S *SSS S S S 55 S 0. 000 a 1 i i INTERNATIONAL SEARCH REPORT International Application No PCT/AU 86/00060 1. CLASSIFICATION OF SUBJECT MATTER (it several classification symools aonly, indicate all) I According to Internat nal Patent Classification (IPC) or to both National Classification and IPC Int. Cl. C07K 15/06, 15/12, 3/20, C12N 15/00, G01H 33/577 II. FIELDS SEARCHED Minimum Documentation Searched 7 Classificatlon System Classification Symbols IPC C07G 7/00, C07K 15/06 US Cl. 260-112 R Documentation Searched other than Minimum Documentation to the Extent thur such Documents are Included In the Fields Searched AU C07G 7/00 (010, 011, 014, 101), C07K 15/06 III. DOCUMENTS CONSIDERED TO BE RELEVANT' Category Citation of Document, i1 with Indication, where apo;ropriate, of the relevant passages 12 Relevant to Claim No. 1 X Journal of Reproduction and 'Fertility, Volume 71, 1-12 Number 2, issued 1984 CUniversity of Queensland, Australia), Alice C. Cavanagh, Production in Vitro of Mouse Early Pregnancy Factor and Purification to Homogeneity, see pages 581-92 X,Y Reproductive Immunology, Proceedings of 2nd 1-12,25 International Congress, Published 1983 by Elsevier Science Publishers (Amsterdam), Studies on Human Early Pregnancy Factor, pages 157-69, by Timothy K. Roberts and Cheng Y. Smart X Pregnancy Proteins, Biological, Chemical Clinical 1-12 Applications, Published 1982 by Academic Press (Australia) (Conference Proceeding), Frank Clark et al, Biochemistry of Early Pregnancy Factor, pages 407-412 X Journal of Reproductive Immunology, Volume 5 1-12 Number 5 1983 (Griffith Univ., Australia) Shann Wilson et al, In Search of Early Pregnancy Factor isolation of active polypeptides from pregnant ewe's sera, pages 275-86 CONTINUED Special categories of cited documents; 1o later document published after the International fling date document defining the general state of the art which Is not or priority date and not in conflict with the application but considered to be of artcular relevance cited to understand the principle or theory underlying the invention earlier document but published on or after the International document of particular relevance: the claimse invention filing date cannot be considered novel or cannot be conialered to document which may throw doubts on priority claim(s) or involve an inventive step which is cited to establish the publication date of another d o ti r r t clam nnin citation or other special reason (as specified) document of particular relevance the claimsl invention cannot be considered to Involve an Inventive step when the document referring to an oral disclosure, use, exhibition or document iS combined with one or more other such docu. other means ments, such combination being obvious to a person skilled document published prior to the International filing date but In the art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search ODte of Mailing o this International Search Report June 1986 (10.06.86) q 7 .o e /7 SLIAE /915 International Searching Authority Silnatoe,9lA )thlixed Officer Australian Patent Office J.P. PULVIRENTI Form PCT/ISA/210 (second sheet) (January 1915) II_ _II~ Interntlonal Aplication No. PCT/AU 86/00060 III. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) Category Citaton of Document, with indication, w ere aPropriate. of te relevant passages Relevant to Claim No X i Journal of Reproductive Immunology, Volume 6 1-12 Number 4 1984 (Griffith Univ., Australia) Shann Wilson et al, In Search of Early Pregnancy Factor characterization of active polypeptides isolated from pregnant ewe's serum, pages 253-60 X Pregnancy Proteins: Biological, Chemical Clinical 1 Applications, Published 1982 by Academic Press (Australia) (Conference Proceeding) Halle Morton et al, Early Pregnancy Factor biology and clinical significance, pages 391-405 Y GB,A, 1563299 (RAFA LABORATORIES LTD) 26 March 1980 (26.03.80) A Journal of Reproduction and Fertility, Volume 69, Number 2, 1983 (Institut Fur Tierzucht und Tieverhalten, Neustadt, West Germany) Halle Morton et al, The Appearance and Characteristics of Early Pregnancy Factor in the Pig, pages 437-46 Form PCT ISA 210 (extra sheet) (January O19l6 ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL APPLICATION NO. PCT/AU 86/ 00060 This Annex lists the known publication level patent family members relating to the patent documents cited in the above-mentioned international search report. The Australian Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent Document Cited in Search Report Patent Family Members GB 1563299 AT 9621/76 AU 20495/76 BE 849667 DE 2657292 ES 45442 FI 763647 FI 763647 FR 2336113 IL 48741 IL 48741 IT 1068738 JP 52082892 NL 7614359 SE 7614387 END OF ANNEX