AU2013202580A1 - Antibodies to HtrA3 - Google Patents

Abstract

The invention provides monoclonal antibodies (mAb) and antigen binding fragments thereof that are, or bind competitively with, an antibody produced from hybridoma cell lines generated using 5 HtrA3 long and short isoforms. Such antibody specifically binds HtrA3 but not HtrA1 or HtrA2. The invention also provides assays utilising the monoclonal antibodies and the use of such assays for detecting a disease involving dysregulation of HtrA3, such as preeclampsia and cancer. The monoclonal antibodies may also be used to treat cancer or preeclampsia or other diseases involving dysregulation of HtrA3. 4223871_1 (GHMatters) P89027.AU.1

Description

1 ANTIBODIES TO HTRA3 Field The invention relates to antibodies to HtrA3 and methods, assays and kits using such 5 antibodies to determine susceptibility to preeclampsia. Background The high temperature requirement A (HtrA) proteases are a well conserved family of serine proteases identified in organisms ranging from bacteria to mammals. HtrAs are known to 10 have important functions in protecting cells from stress conditions such as heat shock, oxidative stress, inflammation, ischemia/reperfusion and cancer. To date, there are four mammalian HtrA homologues identified. The first three members (HtrAl, HtrA2/Omi, HtrA3) have been cloned and investigated for expression and function. The fourth HtrA (HtrA4) is relatively less characterised. 15 HtrA3 was initially identified in the developing placenta both in the mouse and human as a serine protease associated with pregnancy. HtrA3 is now known to inhibit trophoblast invasion during placental development, and to regulate ovarian development, granulosa cell differentiation and luteinisation. Studies in mice have also suggested that HtrA3 inhibits TGF-p signalling during embryo development. 20 HtrA3 has two isoforms [long (HtrA3-L) and short (HtrA3-S)] resulting from alternative mRNA splicing (Figure 1). Full length human HtrA3-L and HtrA3-S contain 453 amino acids (aa) and 357 aa respectively (Figure 1). Both isoforms contain a signature trypsin-like serine protease domain following an N-terminal insulin-like growth factor binding (IGFB) domain and a Kazal protease-inhibitor domain. The HtrA3-L isoform differs from HtrA3-S with the presence of 25 a C-terminal PDZ (postsynaptic density of 95kDa, Discs large and zonula occludens) domain (Figure 1). HtrA3-S thus presents a naturally occurring HtrA lacking the C-terminal PDZ domain. The HtrA3 protein shares 95% similarity between mice and humans, and is expressed more abundantly in the heart, ovary, testis and placenta than other tissues in the mouse. While HtrA3-L is the predominant isoform expressed in the mouse, both HtrA3-L and HtrA3-S are 30 expressed in human tissues, especially in the placenta. It is unknown whether HtrA3-L and HtrA3-S are biochemically distinct, but the PDZ domain in HtrA3-L is predicted to regulate substrate specificity and possibly cellular localisation. Dysregulation of HtrA3 is associated with the development of a number of diseases including cancer and preeclampsia (PE). HtrA3 is downregulated in lung cancers through 4223871_1 (GHMatters) P89027.AU.1 2 smoking-induced DNA methylation and this downregulation increases cancer cell longevity. HtrA3 downregulation in lung cancer also contributes to resistance to chemotherapeutic treatments such as etoposide and cisplatin. HtrA3 is reported to inhibit TGF-p signalling in the endometrium, and is suggested to be 5 involved in ovarian cell homeostasis and tumourigenesis. Downregulation of HtrA3 is associated with the progression of endometrial and ovarian cancer. HtrA3 is thus proposed to be a tumour suppressor and a potential therapeutic aid in cancer treatment. Down regulation of HtrA3 may be predictive of cancer, particularly lung, ovarian and endometrial cancer. At the other end of the spectrum, sustained expression of HtrA3 in the first trimester of 10 pregnancy is associated with the development of preeclampsia (PE). PE is a pregnancy-specific multisystemic disorder primarily involving hypertension and proteinuria. PE is estimated to occur in 0.4 to 7% of live births worldwide and is responsible for 14% of pregnancy related mortalities. Women in developing countries with reduced access to medical treatment in particularly, face a higher incidence of PE related morbidity and mortality compared to those in developed 15 countries. PE occurs in previously normotensive women after 20 weeks of gestation. Abnormal first trimester placentation, generating a high state of maternal inflammation, precedes the onset of PE. PE often restricts blood flow to the foetus causing intrauterine growth restriction (IUGR). As symptoms of PE only resolve once the placenta is removed, the present "cure" for PE is 20 premature delivery of the baby and the placenta. Aside from complications resulting from premature birth, babies born from a PE pregnancy have a higher risk of developing chronic diseases (endocrine, nutritional and metabolic diseases, as well as stroke and hypertension) in adulthood. Women who have had a PE pregnancy also have a higher risk for cardiovascular disease later in life. 25 Early diagnosis of PE is critical for timely clinical intervention and supportive management of pregnant mothers and their foetuses. However, to date there is no clinically useful biochemical diagnosis that can predict PE in early pregnancy. As persistently high serum levels of HtrA3 are detected at the end of the first trimester in pregnant women who subsequently develop PE, we have proposed that monitoring HtrA3 in maternal blood during 30 early pregnancy would identify women at risk for PE. However to date reagents for detecting HtrA3 in serum have not been available. It is an aim of a preferred embodiment of the present invention to provide an agent suitable for 4223871_1 (GHMatters) P89027.AU.1 3 monitoring HtrA3 in blood or serum or plasma or urine so that such an agent may be used in the diagnosis and treatment of diseases involving HtrA3 dysregulation, particularly PE. Summary 5 The development and characterisation of a panel of highly specific HtrA3 monoclonal antibodies (mAbs) is described herein. These mAbs were epitope-mapped and tested for a range of applications including western blotting and immunohistochemistry. The mAbs were used to develop highly sensitive and high-throughput assays to detect HtrA3 in human serum. After being validated on recombinant HtrA3 proteins, the optimised assays were applied to first 10 trimester serum samples collected from women who subsequently developed PE later in pregnancy, which detected significantly higher levels of HtrA3 compared to gestation-matched controls. This result demonstrates the critical importance of these HtrA3 mAbs and the newly developed HtrA3 serum detection assays for the diagnosis of PE and other diseases associated with HtrA3 dysregulation. 15 The present invention in a first aspect provides a monoclonal antibody (mAb) and antigen binding fragment thereof that is, or binds competitively with, an antibody produced from hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019, hybridoma cell line 3E6 deposited with CellBank Australia as accession number 20 CBA20120017, hybridoma cell line 2C4 deposited with CellBank Australia as accession number CBA20120015 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018. Such antibody specifically binds HtrA3 but not HtrAl or HtrA2 and optionally is neutralising for an HtrA3 activity. Such antibodies were all deposited on 13 January 2012. 25 The present invention in a second aspect provides a monoclonal antibody that binds a HtrA3 short isoform and a HtrA3 long isoform but not HtrAl or HtrA2, in which the antibody is produced by hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019 and hybridoma cell line 2C4 deposited with CellBank Australia as accession 30 number CBA20120015. The present invention in a third aspect provides a monoclonal antibody that binds a HtrA3 long isoform but not a HtrA3 short isoform or HtrAl or HtrA2, in which the antibody is produced hybridoma cell line 3E6 deposited with CellBank Australia as accession number 4223871_1 (GHMatters) P89027.AU.1 4 CBA20120017 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018. The present invention in a fourth aspect provides an antagonist monoclonal antibody for HtrA3, which antibody is produced from hybridoma cell line 1OH10 deposited with CellBank 5 Australia as accession number CBA20120016. In one embodiment the antagonist antibody is neutralising for HtrA3. The present invention in a fifth aspect provides an agonist antibody monoclonal antibody for HtrA3, which antibody is produced from hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018. 10 The invention in a sixth aspect provides a monoclonal antibody produced by hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019, hybridoma cell line 3E6 deposited with CellBank Australia as accession number CBA20120017, hybridoma cell line 2C4 deposited with CellBank Australia as accession number CBA20120015 15 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018, conjugated to acceptor beads. The invention in a seventh aspect provides a biotinylated monoclonal antibody produced by hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number 20 CBA20120019, hybridoma cell line 3E6 deposited with CellBank Australia as accession number CBA20120017, hybridoma cell line 2C4 deposited with CellBank Australia as accession number CBA20120015 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018 The invention in an eighth aspect provides a kit comprising a monoclonal antibody or 25 antigen binding fragment thereof that specifically binds to the same epitope as a monoclonal antibody produced by hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019, hybridoma cell line 3E6 deposited with CellBank Australia as accession number CBA20120017, hybridoma cell line 2C4 deposited with CellBank Australia as 30 accession number CBA20120015 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018. The present invention in a ninth aspect provides a kit for HtrA3 short isoform and a HtrA3 long isoform but not HtrA1 or HtrA2, the kit comprising an antibody produced by 4223871_1 (GHMatters) P89027.AU.1 5 hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019 and hybridoma cell line 2C4 deposited with CellBank Australia as accession number. 5 The present invention in a tenth aspect provides a kit for HtrA3 long isoform but not a HtrA3 short isoform or HtrA1 or HtrA2, the kit comprising an antibody produced hybridoma cell line 3E6 deposited with CellBank Australia as accession number CBA20120017and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018. In some embodiments of the eighth to tenth aspects, the kit provides as an enzyme 10 linked immunosorbent assay (ELISA) and more particularly an AlphaLISA. In one embodiment the kit provides pairs of the monoclonal antibodies and particularly 1OH10 paired with 2C4 and 1OH10 paired with 6G6. In some embodiments of the eighth to tenth aspects, the kit provides as an enzyme linked immunosorbent assay (ELISA) and more particularly a sandwich ELISA. In one 15 embodiment the kit provides pairs of the monoclonal antibodies and particularly 1OH10 as the capture antibody paired with 9C9 as the detection antibody, and 1OH10 as the capture antibody paired with 6G6 as the detection antibody. The invention in an eleventh aspect relates to use of a monoclonal antibody or antigen binding fragment thereof that specifically binds to the same epitope as a monoclonal antibody 20 produced by hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019, hybridoma cell line 3E6 deposited with CellBank Australia as accession number CBA20120017, hybridoma cell line 2C4 deposited with CellBank Australia as accession number CBA20120015 and hybridoma cell line 6G6 deposited with CellBank Australia as accession 25 number CBA20120018 for detecting the presence or amount of HtrA3 in a biological sample. The invention in a twelfth aspect relates to use of a monoclonal antibody or antigen binding fragment thereof that specifically binds to the same epitope as a monoclonal antibody produced by hybridoma cells line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession 30 number CBA20120019, hybridoma cell line 3E6 deposited with CellBank Australia as accession number CBA20120017, hybridoma cell line 2C4 deposited with CellBank Australia as accession number CBA20120015 and hybridoma cell line 6G6 deposited with CellBank Australia as 4223871_1 (GHMatters) P89027.AU.1 6 accession number CBA20120018 in the diagnosis of diseases involving dysregulation of HtrA3 such as preeclampsia and cancer, particularly lung cancer. The invention in a thirteenth aspect provides an assay for detecting HtrA3 in a serum or plasma or blood or urine sample, the method comprising contacting the sample with at least one 5 antibody or fragment thereof produced by hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019, hybridoma cell line 3E6 deposited with CellBank Australia as accession number CBA20120017, hybridoma cell line 2C4 deposited with CellBank Australia as accession number CBA20120015 and hybridoma cell line 6G6 deposited with 10 CellBank Australia as accession number CBA20120018 and detecting binding of the antibody or fragment thereof to HtrA3. In some embodiments, this method includes the use of at least two different antibodies or fragments thereof described herein. In some embodiments, the assay comprises an enzyme- linked immunosorbent assay (ELISA) and more particularly an AlphaLISA. 15 The invention in a fourteenth aspect provides a method for detecting a disease involving dysregulation of HtrA3, the method comprising assaying for HtrA3 using the assay of the thirteenth aspect. The invention in a fifteenth aspect provides a method of treating cancer comprising administering an agonist antibody to HtrA3 which antibody is produced from hybridoma cell line 20 6G6 deposited with CellBank Australia as accession number CBA20120018. In an alternative form the fifteenth aspect provides an antibody produced from hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018 for treating cancer or use of an antibody produced from hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018 in the manufacture of a medicament for 25 treating cancer. In an embodiment of the fifteenth aspect the cancer is but not limited to lung cancer, ovarian cancer or endometrial cancer. The invention in a sixteenth aspect provides a method of treating preeclampsia or other diseases involving dysregulation of HtrA3 comprising administering an antagonist antibody to 30 HtrA3 which antibody is produced from hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016. In an alternative form the sixteenth aspect provides an antibody produced from hybridoma cell line 1OH10 deposited with CellBank Australia as accession number 4223871_1 (GHMatters) P89027.AU.1 7 CBA20120016 for treating preeclampsia or other diseases involving dysregulation of HtrA3, or use of an antibody produced from hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016in the manufacture of a medicament for treating preeclampsia or other diseases involving dysregulation of HtrA3. 5 In an embodiment of the sixteenth aspect the antibody is neutralising for HtrA3. Also provided is an isolated nucleic acid molecule encoding any of the antibodies described herein, a vector comprising the isolated nucleic acid molecule, a host cell transformed with the nucleic acid molecule, and a method of producing the antibody comprising culturing the host cell under conditions wherein the nucleic acid molecule is expressed to produce the 10 antibody and optionally recovering the antibody from the host cell. In some embodiments, the antibody or fragment thereof is chimeric or is humanised. In some embodiments, the fragment is selected from the group of: a Fab fragment, a F(ab') 2 fragment, and a scFv fragment. In some embodiments, the antibody or fragment thereof is glycosylated. In some embodiments, the antibody or fragment thereof contains one or more 15 complementary determining regions of the light or heavy chain of the antibody produced by hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019, hybridoma cell line 3E6 deposited with CellBank Australia as accession number CBA20120017, hybridoma cell line 2C4 deposited with CellBank Australia as accession number 20 CBA20120015 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018 or an antigen-binding fragment of any one or more thereof. The invention also provides a method of producing a hybridoma cell line that expresses a binding protein for HtrA3 but not HtrA1 or HtrA2 comprising the steps of: a) immunising a BALB/C mouse with an antigen comprising HtrA3-L-S305A or amino acids 230-239 of HtrA3-L 25 and HtrA3-S (TIKIHPKKKL - SEQ ID NO: 4) for a time and under conditions sufficient for the mouse to produce antibodies against the antigen; b) harvesting and purifying cells from the spleen of the mouse; c) fusing the spleen cells with myeloma cells in order to produce hybridomas; and d) selecting a hybridoma cell line which expresses the binding protein which bind either or both of HtrA3-L and HtrA3-S but not HtrA1 or HtrA2. In this method, the 30 hybridoma cell line can be the cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019, hybridoma cell line 3E6 deposited with CellBank Australia as accession number CBA20120017, hybridoma cell line 2C4 deposited with CellBank Australia as accession 4223871_1 (GHMatters) P89027.AU.1 8 number CBA20120015 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018. Also provided are cells of the hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank 5 Australia as accession number CBA20120019, hybridoma cell line 3E6 deposited with CellBank Australia as accession number CBA20120017, hybridoma cell line 2C4 deposited with CellBank Australia as accession number CBA20120015 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018 Also provided is a method of determining whether to discharge an inpatient or to initiate 10 or continue monitoring or treatment of a subject for preeclampsia on an inpatient basis including obtaining a sample from a subject and determining the level of HtrA3 in the sample using at least one antibody or fragment described herein, where an elevated level of HtrA3 in the sample compared to a reference level of HtrA3 indicates that inpatient monitoring or treatment for preeclampsia should be initiated or continued. 15 Also provided are methods of selecting a subject for participation in a clinical study including obtaining a sample from a subject, determining the level of HtrA3 in the sample using at least one antibody or fragment thereof described herein, and selecting the subject for participation in a clinical study if the subject's level of HtrA3 relative to a reference level of HtrA3 indicates that the subject should be selected for participation in a clinical study. In some 20 embodiments, the presence of an elevated level of HtrA3 indicates that the subject should be selected for participation in a clinical study. Also provided are methods for selecting a therapeutic treatment for a subject including determining the level of HtrA3 in a biological sample from the subject using at least one antibody or fragment thereof described herein, wherein the subject's level of HtrA3 relative to a 25 reference level of HtrA3 is used to select a therapeutic treatment for the subject. In some embodiments, the presence of an elevated level of HtrA3 is used to select the therapeutic treatment for the subject. In one embodiment the treatment is in accordance with the fifteenth or sixteenth aspects. 30 In some embodiments the inpatient or subject has one or more risk factors for preeclampsia. Preeclampsia develops only during pregnancy. Risk factors include history of preeclampsia, first pregnancy, new paternity, age, obesity, multiple pregnancies with different partners, prolonged interval between pregnancies, diabetes and gestational diabetes, history of 4223871_1 (GHMatters) P89027.AU.1 9 certain conditions such as chronic high blood pressure, migraine headaches, diabetes, kidney disease, rheumatoid arthritis or lupus, having other health conditions such as both urinary tract infections and periodontal disease or vitamin D insufficiency. In some embodiments the inpatient or subject has one or more symptoms of 5 preeclampsia, such as hypertension and proteinuria. In some embodiments the inpatient or subject has high blood pressure (two separate readings taken at least six hours apart of 140 or more in systolic blood pressure and/or 90 or more in diastolic blood pressure) and/or 300 mg of protein in a 24-hour urine sample Unless otherwise defined, all technical and scientific terms used herein have the same 10 meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention. Other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are 15 incorporated by reference in their entirety. Detailed Description The present invention relates to monoclonal antibodies and their use in methods or kits to detect HtrA3 and thus diagnose disorders involving HtrA3 dysregulation such as some 20 cancers and preeclampsia. HtrA3 has two protein isoforms [long (HtrA3-L) and short (HtrA3-S)] resulting from alternative mRNA splicing (Figure 1). Full length human HtrA3-L and HtrA3-S contain 453 amino acids (aa) and 357 aa respectively. The protein, isoforms and its association with preeclampsia was first suggested in 2003 (Nie GY et al., 2003 Biochem J 371:39-48, Nie GY et 25 al., 2003 Mol Hum Reprod 9:279-290) and validated in 2011 (Li et al., 2011 J Clin Endocrinol Metab 96: 403-411). By the term HtrA3 is meant a protein containing a sequence at least 90% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to NCBI Accession No. P83110-2 (HtrA3-L) SEQ ID NO: 1 30 4223871_1 (GHMatters) P89027.AU.1 10 10 20 30 40 50 60 MQARALLLAA LAALALAREP PAAPCPARCD VSRCPSPRCP GGYVPDLCNC CLVCAASEGE 70 80 90 100 110 120 PCGGPLDSPC GESLECVRGL CRCRWSHAVC GTDGHTYANV CALQAASRRA LQLSGTPVRQ 5 130 140 150 160 170 180 LQKGACPLGL HQLSSPRYKF NFIADVVEKI APAVVHIELF LRHPLFGRNV PLSSGSGFIM 190 200 210 220 230 240 SEAGLIITNA HVVSSNSAAP GRQQLKVQLQ NGDSYEATIK DIDKKSDIAT IKIHPKKKLP 250 260 270 280 290 300 10 VLLLGHSADL RPGEFVVAIG SPFALQNTVT TGIVSTAQRE GRELGLRDSD MDYIQTDAII 310 320 330 340 350 360 NYGNSGGPLV NLDGEVIGIN TLKVTAGISF AIPSDRITRF LTEFQDKQIK DWKKRFIGIR 370 380 390 400 410 420 MRTITPSLVD ELKASNPDFP EVSSGIYVQE VAPNSPSQRG GIQDGDIIVK VNGRPLVDSS 15 430 440 450 ELQEAVLTES PLLLEVRRGN DDLLFSIAPE VVM (SEQ ID NO: 1 ) or to NCBI Accession No. AAP42283.1 (HtrA3-S ) SEQ ID NO: 2 20 10 20 30 40 50 60 MQARALLLAA LAALALAREP PAAPCPARCD VSRCPSPRCP GGYVPDLCNC CLVCAASEGE 70 80 90 100 110 120 PCGGPLDSPC GESLECVRGL CRCRWSHAVC GTDGHTYANV CALQAASRRA LQLSGTPVRQ 25 130 140 150 160 170 180 LQKGACPLGL HQLSSPRYKF NFIADVVEKI APAVVHIELF LRHPLFGRNV PLSSGSGFIM 190 200 210 220 230 240 SEAGLIITNA HVVSSNSAAP GRQQLKVQLQ NGDSYEATIK DIDKKSDIAT IKIHPKKKLP 250 260 270 280 290 300 30 VLLLGHSADL RPGEFVVAIG SPFALQNTVT TGIVSTAQRE GRELGLRDSD MDYIQTDAII 310 320 330 340 350 NYGNSGGPLV NLDGEVIGIN TLKVTAGISF AIPSDRITRF LTEFQDKQIK APSLAVH (SEQ ID NO: 2) or a nucleic acid containing a sequence at least 90% identical (e.g., at least 95%, 96%, 97%, 35 98%, 99%, or 100%) identical) to NCBI Accession No. NM053044 (SEQ ID NO: 3):. 42238711 (GHMatters) P89027.AU.1 11 1 ggagggagct ggtccctgcg ctccctgcgc cctggggatg cccctgccgc cctgacgccc 61 gccagcctga gccaccggcg catgtgaccg cgcgtccgcc ccagtcccat ccgtaggcgc 121 ccggcgcccg gocccgcagc ggcctcgttg tccccgccgg cccccgcccg gtctcccgcg 181 ctgccacccg ccgccggccc tgccgccatg caggcgcgag cgctgctcct ggccgcgttg 5 241 gccgcgctgg cgctggcccg ggagccccct gcggcgccgt gtcccgcgcg ctgcgacgtg 301 tcgcggtgtc ccagcccccg ctgccccggc ggctacgtgc ccgacctctg caactgctgc 361 ctggtgtgcg ccgccagcga gggcgagccc tgtggcggcc ctctggactc gccttgcggc 421 gagagcctgg agtgcgtgcg cggcctatgc cgctgccgct ggtcgcacgc cgtgtgtggc 481 accgacgggc acacctatgc caacgtgtgc gcgctgcagg cggccagccg ccgcgcgctg 10 541 cagctctccg ggacgcccgt gcgccagctg cagaagggcg cctgcccgtt gggtctccac 601 cagctgagca gcccgcgcta caagttcaac ttcattgctg acgtggtgga gaagatcgca 661 ccagccgtgg tccacataga gctcttcctg agacacccgc tgtttggccg caacgtgccc 721 ctgtccagcg gttctggctt catcatgtca gaggccggcc tgatcatcac caatgcccac 781 gtggtgtcca gcaacagtgc tgccccgggc aggcagcagc tcaaggtgca gctacagaat 15 841 ggggactcct atgaggccac catcaaagac atcgacaaga agtcggacat tgccaccatc 901 aagatccatc ccaagaaaaa gctccctgtg ttgttgctgg gtcactcggc cgacctgcgg 961 cctggggagt ttgtggtggc catcggcagt cccttcgccc tacagaacac agtgacaacg 1021 ggcatcgtca gcactgccca gcgggagggc agggagctgg gcctccggga ctccgacatg 1081 gactacatcc agacggatgc catcatcaac tacgggaact ccgggggacc actggtgaac 20 1141 ctggatggcg aggtcattgg catcaacacg ctcaaggtca cggctggcat ctcctttgcc 1201 atcccctcag accgcatcac acggttcctc acagagttcc aagacaagca gatcaaagac 1261 tggaagaagc gcttcatcgg catacggatg cggacgatca caccaagcct ggtggatgag 1321 ctgaaggcca gcaacccgga cttcccagag gtcagcagtg gaatttatgt gcaagaggtt 1381 gcgccgaatt caccttctca gagaggcggc atccaagatg gtgacatcat cgtcaaggtc 25 1441 aacgggcgtc ctctagtgga ctcgagtgag ctgcaggagg ccgtgctgac cgagtctcct 1501 ctcctactgg aggtgcggcg ggggaacgac gacctcctct tcagcatcgc acctgaggtg 1561 gtcatgtgag gggcgcattc ctccagcgcc aagcgtcaga gcctgcagac aacggagggc 1621 agcgcccccc cgagatcagg acgaaggacc accgtcggtc ctcagcaggg cggcagcctc 1681 ctcctggctg tccggggcag agcggaggct gggcttggcc aggggcccga atttccgcct 30 1741 ggggagtgtt ggatccacat cccggtgccg gggagggaag cccaacatcc ccttgtacag 1801 atgatcctga aagtcacttc caagttctcc ggatattcac aaaactgcct tccatggagg 1861 tcccctcctc tcctagcttc ccgcctctgc ccctgtgaac acccatctgc agtatcccct 1921 gctcctgccc ctcctactgc aggtctgggc tgccaagctt cttcccccct gacaaacgcc 1981 cacctgacct gaggccccag cttccctctg ccctaggact taccaagctg tagggccagg 35 2041 gctgctgcct gccagcctgg ggtccctgga ggacaggtca catctgatcc ctttggggtg 2101 cgggggtggg gtccagccca gagcaggcac tgagtgaatg ccccctggct gcggagctga 2161 gccccgccct gccatgaggt tttcctcccc aggcaggcag gaggccgcgg ggagcacgtg 2221 gaaagttggc tgctgcctgg ggaagcttct cctccccaag gcggccatgg ggcagcctgc 2281 agaggacagt ggacgtggag ctgcggggtg tgaggactga gccggcttcc ccttcccacg 40 2341 cagctctggg atgcagcagc cgctcgcatg gaagtgccgc ccagaggcat gcaggctgct 2401 gggcaccacc ccctcatcca gggaacgagt gtgtctcaag gggcatttgt gagctttgct 2461 gtaaatggat tcccagtgtt gcttgtactg tatgtttctc tactgtatgg aaaataaagt 2521 ttacaagcac acggttctca gccaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2581 aaaaaaaaa (SEQ ID NO: 3) 45 By the term "elevated" or "elevation" is meant a difference, e.g., a statistically significant difference (e.g., an increase) in a determined or measured level (e.g., a HtrA3 protein level) compared to a reference level (e.g., a level of HtrA3 in a subject not having PE, a subject not presenting with two or more symptoms of PE, or a subject not identified as being at risk of 4223871_1 (GHMatters) P89027.AU.1 12 developing PE, or a threshold level of HtrA3). In some embodiments, the reference is a threshold level, and any level above that is considered "elevated." Additional reference levels of HtrA3 are described herein. Given their ability to bind to HtrA3 or epitopes or portions thereof, the antibodies as 5 disclosed herein can be used to detect and/or quantify an amount of HtrA3 in a biological sample (such as, for example, serum, blood, tissue or plasma), using a conventional competitive or non-competitive immunoassay (e.g., an enzyme linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), immunometric sandwich assay or tissue immunohistochemistry). Such detection may then result in a diagnosis of PE or cancer for the 10 patient from which the biological sample was obtained. Suitable detectable substances for labelling the antibody include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, p galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes 15 include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a radionuclides (e.g. 3 H, 14C, 35 s, 90 Y, 99 Tc, 1 'in, 1251 1311 177 Lu, 166 Ho or 153 Sm). As an alternative to labelling the antibody, the antigen can be assayed in biological fluids 20 by a competition immunoassay utilising recombinant standards labelled with a detectable substance and an unlabelled antibody. In this assay, the biological sample, the labelled recombinant antigen standard and the antibody are combined, and the amount of labelled peptide standard bound to the unlabelled antibody is determined. The amount of antigen in the biological sample is inversely proportional to the amount of labelled antigen standard bound to 25 the antibody. In this method, the antibody can comprise an antigen-binding domain that binds to HtrA3 such as in monoclonal antibody produced by a hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019, hybridoma cell line 3E6 deposited with CellBank Australia as accession number CBA20120017, hybridoma cell line 2C4 deposited with 30 CellBank Australia as accession number CBA20120015 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018 or an antigen-binding fragment of any one or more thereof. 4223871_1 (GHMatters) P89027.AU.1 13 To illustrate the above assays, an antibody to HtrA3 (epitopes or portions of full length HtrA3), such as an antibody produced by a hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019, hybridoma cell line 3E6 deposited with CellBank 5 Australia as accession number CBA20120017, hybridoma cell line 2C4 deposited with CellBank Australia as accession number CBA20120015 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018 is for example coated on a solid phase (or is present in a liquid phase). The test or biological sample (e.g., serum, plasma, urine, etc.) is then contacted with the solid phase. If HtrA3 is present in the sample, the antibody bound to 10 the solid phase will bind to the HtrA3 which may then be detected by either a direct or indirect method. The direct method comprises simply detecting presence of the complex itself and thus presence of the HtrA3. In the indirect method, a conjugate is added to the bound HtrA3. The conjugate comprises a second antibody (different from the first antibody coated onto the solid phase), which binds to the bound HtrA3 antigen, attached to a signal generating compound or 15 label. Should the second antibody bind to the bound antigen, the signal-generating compound generates a measurable signal. Such signal then indicates presence of the antigen in the test sample. It should be noted that the initial capture antibody (for detecting HtrA3) used in the immunoassay may be covalently or non-covalently (e.g., ionic, hydrophobic, etc.) attached to the solid phase. Linking agents for covalent attachment are known in the art and may be part of 20 the solid phase or derivatised to it prior to coating. Examples of solid phases used in diagnostic immunoassays are porous and non-porous materials, latex particles, magnetic particles, microparticles (see U.S. Pat. No. 5,705,330), beads, membranes, microtiter wells and plastic tubes. The choice of solid phase material and method of labelling the antigen or antibody present in the conjugate, if desired, are determined 25 based upon desired assay format performance characteristics As noted above, the conjugate (or indicator reagent) will comprise an antibody (or anti antibody, depending upon the assay), attached to a signal-generating compound or label. This signal-generating compound or "label" is itself detectable or may be reacted with one or more additional compounds to generate a detectable product. Examples of signal-generating 30 compounds include chromogens, radioisotopes (e.g., 1251, 1311, 32P, 3H, 35S and 14C), chemiluminescent compounds (e.g., acridinium), particles (visible or fluorescent), nucleic acids, complexing agents, or catalysts such as enzymes (e.g., alkaline phosphatase, acid phosphatase, horseradish peroxidase, beta-galactosidase and ribonuclease). In the case of 4223871_1 (GHMatters) P89027.AU.1 14 enzyme use (e.g., alkaline phosphatase or horseradish peroxidase), addition of a chromo-, fluoro-, or lumo-genic substrate results in generation of a detectable signal. Other detection systems such as time-resolved fluorescence, internal-reflection fluorescence, amplification (e.g., polymerase chain reaction) and Raman spectroscopy are also useful. 5 Alternatively, in order to detect the presence of HtrA3 in a biological sample, one may coat the solid phase with HtrA3 and then contact the solid phase with labelled antibody to HtrA3, such as monoclonal antibody produced by a hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019, hybridoma cell line 3E6 deposited with 10 CellBank Australia as accession number CBA20120017, hybridoma cell line 2C4 deposited with CellBank Australia as accession number CBA20120015 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018, for a time and under conditions sufficient to allow the immobilised antigen to bind to the labelled antibody. Subsequent thereto, the test sample may be added to the antigen-antibody complex. If HtrA3 is present in the test 15 sample, it will then bind to the bound labelled antibody. A detectable signal is then generated by the label indicating presence of the HtrA3 in the test sample. Additionally, in an alternative assay format, one may use a HtrA3 recombinant standard labelled with a detectable substance and an unlabelled antibody such as produced by a hybridoma cell line 1OH10 deposited with CellBank Australia as accession number 20 CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019, hybridoma cell line 3E6 deposited with CellBank Australia as accession number CBA20120017, hybridoma cell line 2C4 deposited with CellBank Australia as accession number CBA20120015 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018. In this assay, the biological test sample, the labelled recombinant HtrA3 25 standard and the monoclonal antibody are combined, and the amount of labelled HtrA3 standard bound to the unlabelled antibody is determined. The amount of HtrA3 in the biological sample is inversely proportional to the amount of labelled HtrA3 standard bound to the antibody. Other assay formats which may be used for purposes of the present disclosure, in order to simultaneously detect antigens and antibodies include, for example, Dual assay strip blots, a 30 rapid test, a western blot, as well as the use of paramagnetic particles in, for example, an Architect@ assay (Frank Quinn, The Immunoassay Handbook, Second edition, edited by David Wild, pages 363-367, 2001). Such formats are known to those of ordinary skill in the art. 4223871_1 (GHMatters) P89027.AU.1 15 It should also be noted that the elements of the assays described above are particularly suitable for use in the form of a kit. The kit may also comprise one container such as vial, bottles or strip, with each container with a pre-set solid phase, and other containers containing the respective conjugates. These kits may also contain vials or containers of other reagents needed for 5 performing the assay, such as washing, processing and indicator reagents. Any of the exemplary formats herein and any assay or kit according to the invention can be adapted or optimised for use in automated and semi-automated systems (including those in which there is a solid phase comprising a microparticle), as described, e.g., in U.S. Pat. Nos. 5,089,424 and 5,006,309, and as, e.g., commercially marketed by Abbott Laboratories (Abbott 10 Park, Ill.) including but not limited to Abbott's ARCHITECT@, AxSYM, IMX, PRISM, and Quantum II platforms, as well as other platforms. Additionally, the assays and kits of the present invention optionally can be adapted or optimised for point of care assay systems, including Abbott's Point of Care (i-STATIm) electrochemical immunoassay system. Immunosensors and methods of manufacturing and operating them in single-use test devices are described, for 15 example in U.S. Pat. No. 5,063,081 and published U.S. Patent Application Nos. 20030170881, 20040018577, 20050054078, and 20060160164 (incorporated by reference herein for their teachings regarding same). In one embodiment HtrA3 is detected in an AlphaLISA assay. The assay comprises contacting a sample with a biotinylated monoclonal antibody produced by hybridoma cell line 20 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019, hybridoma cell line 3E6 deposited with CellBank Australia as accession number CBA20120017, hybridoma cell line 2C4 deposited with CellBank Australia as accession number CBA20120015 or hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018, and 25 AlphaLISA acceptor beads conjugated with a monoclonal antibody produced by a hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019, hybridoma cell line 3E6 deposited with CellBank Australia as accession number CBA20120017, hybridoma cell line 2C4 deposited with CellBank Australia as accession number CBA20120015 30 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018, under conditions for binding to occur, adding streptavidin donor beads and detecting binding. In one embodiment binding is detected by determining europium luminescence. 4223871_1 (GHMatters) P89027.AU.1 16 In one embodiment the monoclonal antibody conjugated to acceptor beads is 1OH 10. In one embodiment the biotinylated monoclonal antibody is 6G6, 2C4 or 3E6 (in order of decreasing reactivity). By the term "health care facility" is meant a location where a subject can receive medical 5 care from a health care professional (e.g., a nurse, a physician, or a physician's assistant). Non limiting examples of health care facilities include hospitals, clinics, and assisted care facilities (e.g., a nursing home). By the term "inpatient" is meant a subject that is admitted to a medical care facility (e.g., a hospital or an assisted care facility). 10 By the term "inpatient treatment" is meant the monitoring and/or medical treatment of a subject that is admitted to a health care facility (e.g., a hospital or assisted care facility). By the term "reference level" is meant a threshold level or a level in a control subject or control patient population. A reference level will depend on the assay performed and can be determined by one of ordinary skill in the art. A reference level can be a baseline level or a level 15 in the same patient measured at an earlier or later point in time. Some non-limiting examples of reference levels of HtrA3 include the level of HtrA3 in a subject that: has not been diagnosed as having PE; does not present with at least two or more symptoms of PE; does not have a family history of PE; does not have proteinuria or does not have hypertension. By the term "therapeutic treatment" or "treatment" is meant the administration of one or 20 more pharmaceutical agents to a subject or the performance of a medical procedure on the body of a subject. The term therapeutic treatment also includes an adjustment (e.g., increase or decrease) in the dose or frequency of one or more pharmaceutical agents that a subject can be taking, the administration of one or more new pharmaceutical agents to the subject, or the removal of one or more pharmaceutical agents from the subject's treatment plan. 25 As used herein, a "subject" is a mammal, e.g., a human. As PE only occurs during pregnancy, for embodiments related to diagnosis or monitoring PE, the subject is a pregnant woman. As used herein, a "biological sample" includes one or more of blood, serum, plasma, urine, and body tissue. Generally, a biological sample is a sample containing serum, blood, or 30 plasma. As used herein, the, term "antibody" refers to a protein that generally contains heavy chain polypeptides and light chain polypeptides. Antigen recognition and binding occurs within the variable regions of the heavy and light chains. Single domain antibodies having one heavy 4223871_1 (GHMatters) P89027.AU.1 17 chain and one light chain, and heavy chain antibodies devoid of light chains, are also known. A given antibody comprises one of five different types of heavy chains, called alpha, delta, epsilon, gamma, and mu, the categorisation of which is based on the amino acid sequence of the heavy chain constant region. These different types of heavy chains give rise to five classes 5 of antibodies, IgA (including IgAl and IgA2), IgD, IgE, IgG (IgGI , IgG2, IgG3, and IgG4) and IgM, respectively. A given antibody also comprises one of two types of light chains, called kappa or lambda, the categorisation of which is based on the amino acid sequence of the light chain constant domains. IgG, IgD, and IgE antibodies generally contain two identical heavy chains and two identical light chains and two antigen combining domains, each composed of a 10 heavy chain variable region (VH) and a light chain variable region (VL). Generally IgA antibodies are composed of two monomers, each monomer composed of two heavy chains and two light chains (as for IgG, IgD, and IgE antibodies). In this way the IgA molecule has four antigen binding domains, each again composed of a VH and a VL. Certain IgA antibodies are monomeric in that they are composed of two heavy chains and two light chains. Secreted IgM 15 antibodies are generally composed of five monomers, each monomer composed of two heavy chains and two light chains (as for IgG and IgE antibodies). In this way the secreted IgM molecule has ten antigen-binding domains, each again composed of a VH and a VL. A cell surface form of IgM also exists and this has a two heavy chain/two light chain structure similar to IgG, IgD, and IgE antibodies. 20 The term "monoclonal antibody" as used herein refers to an antibody (or antibody fragment) obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional 25 (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesised by the hybridoma culture, and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as 30 being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by 4223871_1 (GHMatters) P89027.AU.1 18 recombinant DNA methods. The "monoclonal antibodies" also include clones of antigen recognition and binding-site containing antibody fragments (Fv clones) isolated from phage antibody libraries. The monoclonal antibodies herein specifically include "chimeric" antibodies 5 (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such 10 antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567 to Cabilly et al). As used herein, the term "chimeric antibody" refers to an antibody that has been engineered to comprise at least one human constant region. For example, one or all (e.g., one, two, or three) of the variable regions of the light chain(s) and/or one or all (e.g., one, two, or 15 three) of the variable regions the heavy chain(s) of a mouse antibody (e.g., a mouse monoclonal antibody) can each be joined to a human constant region, such as, without limitation an IgG I human constant region. Chimeric antibodies are typically less immunogenic to humans, relative to non-chimeric antibodies, and thus offer therapeutic benefits in certain situations. Those skilled in the art will be aware of chimeric antibodies, and will also be aware of 20 suitable techniques for their generation. As used herein, the term "fully human antibodies" are antibodies or antigen binding fragments of antibodies that contain only human-derived amino acid sequences. For example, a fully human antibody may be produced from a human B-cell or a human hybridoma cell. In additional embodiments, the antibody may be produced from a transgenic animal that contains 25 the locus for a human heavy chain immunoglobulin and a human light chain immunoglobulin, or contains a nucleic acid that encodes the heavy and light chains of a specific human antibody. "Complementarity-determining region" or "CDR" as the terms are used herein refer to short polypeptide sequences within the variable region of both heavy and light chain polypeptides that are primarily responsible for mediating specific antigen recognition. CDRs have been described 30 by Kabat, et al., J. Biol. Chem. 252, 6609-6616, 1977. There are three CDRs (termed CDR1, CDR2, and CDR3) within each VL and each VH. "Fragment" or "antibody fragment" as the terms are used herein refer to a polypeptide derived from an antibody polypeptide molecule (e.g., an antibody heavy and/or light chain 4223871_1 (GHMatters) P89027.AU.1 19 polypeptide) that does not comprise a full-length antibody polypeptide, but that still comprises at least a portion of a full-length antibody polypeptide that is capable of binding to an antigen. Antibody fragments can comprise a cleaved portion of a full length antibody polypeptide, although the term is not limited to such cleaved fragments. Antibody fragments can include, for 5 example, Fab fragments, F(ab')2 fragments, scFv (single-chain Fv) fragments, linear antibodies, monospecific or multispecific antibody fragments such as bispecific, trispecific, and multispecific antibodies (e.g., diabodies, triabodies, tetrabodies), minibodies, chelating recombinant antibodies, tribodies or bibodies, intrabodies, nanobodies, small modular immunopharmaceuticals (SMIP), binding-domain immunoglobulin fusion proteins, camelised 10 antibodies, and VHH containing antibodies. Additional examples of antigen- binding antibody fragments are known in the art. "Framework region" as the term is used herein refers to amino acid sequences within the variable region of both heavy and light chain polypeptides that are not CDR sequences, and are primarily responsible for maintaining correct positioning of the CDR sequences to permit 15 antigen binding. Although the framework regions themselves typically do not directly participate in antigen binding, as is known in the art, certain residues within the framework regions of certain antibodies can directly participate in antigen binding or can affect the ability of one or more amino acids in CDRs to interact with antigen. "Humanised antibody" as the term is used herein refers to an antibody that has been 20 engineered to comprise one or more human framework regions in the variable region together with non-human (e.g., mouse, rat, or hamster) complementarity- determining regions (CDRs) of the heavy and/or light chain. In some embodiments, a humanised antibody comprises sequences that are entirely human except for the CDR regions. Humanised antibodies are typically less immunogenic to humans, relative to non-humanised antibodies, and thus offer 25 therapeutic benefits in certain situations. Humanised antibodies are known in the art, and suitable techniques for generating humanised antibodies are also known. An antagonist antibody as used herein is an antibody that inhibits the activity of the protein to which it binds. In one embodiment the antibody completely inhibits or neutralises an activity of HtrA3. In one embodiment, the antibody is capable of neutralising at least 60%, or at 30 least 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, still more preferably at least 90%, still more preferably at least 95%, most preferably at least 99% of a biological activity of HtrA3. The activity of HtrA3 that is inhibited may be protease activity. In one embodiment the antibody completely inhibits or neutralises the 4223871_1 (GHMatters) P89027.AU.1 20 protease activity of HtrA3. In one embodiment, the antibody is capable of neutralising at least 60%, or at least 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, still more preferably at least 90%, still more preferably at least 95%, most preferably at least 99% of the protease activity of HtrA3. 5 An agonist antibody as used herein us an antibody that increases the activity of the protein to which it binds. In one embodiment the antibody increases an activity of HtrA3 by at least 10%, or at least 20%, preferably at least 30%, more preferably at least 40%, even more preferably at least 50%, of a biological activity of HtrA3. The activity of HtrA3 that is increased may be protease activity. In one embodiment the antibody is capable of increasing the protease 10 activity of HtrA3 by at least 10%, or at least 20%, preferably at least 30%, more preferably at least 40%, even more preferably at least 50%. An "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the 15 antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under 20 reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. The term "epitope" is used to refer to a binding site for an antibody on a protein. 25 Antibodies which bind to a particular epitope can be identified by "epitope mapping." There are many methods known in the art for mapping and characterising the location of epitopes on proteins, including solving the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays. Competition assays are discussed above and below. According to the gene fragment expression assays, the 30 open reading frame encoding the protein is fragmented either randomly or by specific genetic constructions and the reactivity of the expressed fragments of the protein with the antibody to be tested is determined. The gene fragments may, for example, be produced by PCR and then transcribed and translated into protein in vitro, in the presence of radioactive amino acids. The 4223871_1 (GHMatters) P89027.AU.1 21 binding of the antibody to the radioactively labelled protein fragments is then determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a defined library of overlapping peptide fragments can be tested for 5 binding to the test antibody in simple binding assays. The latter approach is suitable to define linear epitopes of about 5 to 15 amino acids. An antibody binds "essentially the same epitope" as a reference antibody, when the two antibodies recognise identical or sterically overlapping epitopes. The most widely used and rapid methods for determining whether two epitopes bind to identical or sterically overlapping 10 epitopes are competition assays, which can be configured in all number of different formats, using either labelled antigen or labelled antibody. Usually, the antigen is immobilised on a 96 well plate, and the ability of unlabelled antibodies to block the binding of labelled antibodies is measured using radioactive or enzyme labels. The term amino acid or amino acid residue, as used herein, refers to naturally occurring 15 L amino acids or to D amino acids as described further below with respect to variants. The commonly used one- and three-letter abbreviations for amino acids are used herein. "Percent (%) amino acid sequence identity" with respect to the polypeptide sequences referred to herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a sequence, after aligning the sequences and 20 introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the 25 art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and its source code 30 has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, Calif., and the source code for the ALIGN-2 program and instructions for its use are disclosed in International Application 4223871_1 (GHMatters) P89027.AU.1 22 Publication No. W02000/39297 published Jul. 6, 2000. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. For purposes herein, the % amino acid sequence identity of a given amino acid 5 sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y 10 where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically 15 stated otherwise, all % amino acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program. However, % amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2. The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search parameters, wherein all of 20 those search parameters are set to default values including, for example, unmask=yes, strand=all, expected occurrences=1 0, minimum low complexity length=15/5, multi-pass e value=0.01, constant for multi-pass=25, dropoff for final gapped alignment=25 and scoring matrix=BLOSUM62. In situations where NCBI-BLAST2 is employed for amino acid sequence comparisons, 25 the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 30 100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of A and B, and where Y is the 4223871_1 (GHMatters) P89027.AU.1 23 total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. By the term "additional marker" is meant a protein, nucleic acid, lipid, or carbohydrate, or 5 a combination (e.g., two or more) thereof, that is diagnostic of the presence of a particular disease. The methods described herein for diagnosing a subject as having a disease can include detecting a level of HtrA3 and at least one additional marker in a sample from the subject. "Treatment" refers to both therapeutic treatment and prophylactic or preventative 10 measures. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in which the disorder is to be prevented. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilised (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and 15 remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented. In the methods of the invention an antibody can be labelled with a detectable label and 20 the method can include measuring the signal generated by or emitted from the detectable label and detecting the HtrA3 in the biological sample. The detectable label can be a radioactive label, an enzymatic label, a chemiluminescent label, a fluorescence label, a thermometric label, and an immuno-polymerase chain reaction label. The detectable label can be, for example, an acridinium compound. When the detectable label is an acridinium compound, the method may 25 further comprise: a) generating or providing a source of hydrogen peroxide to the antibody antigen complexes; b) adding a basic solution to the mixture of step (a); and c) measuring the light signal generated or emitted in step (b) and detecting HtrA3 in the sample. In the methods of the invention, HtrA3 can be detected in a biological sample by a) contacting the sample with a first monoclonal antibody for HtrA3 as described herein for a time 30 and under conditions sufficient for the formation of first antibody-antigen complexes; b) adding a second antibody to the first antibody/antigen complexes, wherein the second antibody is conjugated to a detectable label, for a time and under conditions sufficient to form first antibody/antigen/second antibody complexes; and c) measuring the signal generated by or 4223871_1 (GHMatters) P89027.AU.1 24 emitted from the detectable label and detecting the HtrA3 in the sample. The first antibody or second antibody can be a monoclonal antibody produced by hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019, hybridoma cell line 3E6 5 deposited with CellBank Australia as accession number CBA20120017, hybridoma cell line 2C4 deposited with CellBank Australia as accession number CBA20120015 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018. In the methods of the invention, HtrA3 can be detected in a biological sample by: a) contacting the sample with 1) a HtrA3 reference antigen, wherein the reference antigen is 10 attached to a detectable label capable of generating a detectable signal and 2) an antibody to HtrA3, for a time and under conditions sufficient to form HtrA3 reference antigen/antibody complexes; b) detecting a signal generated by the detectable label, wherein the amount of HtrA3 antigen detected in the test sample is inversely proportional to the amount of HtrA3 reference antigen bound to the antibody. In the method, the antibody can be a monoclonal 15 antibody produced by hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019, hybridoma cell line 3E6 deposited with CellBank Australia as accession number CBA20120017, hybridoma cell line 2C4 deposited with CellBank Australia as accession number CBA20120015 and hybridoma cell line 6G6 deposited with CellBank 20 Australia as accession number CBA20120018. Persons skilled in the art will appreciate that the methods of the invention cannot be 100% correct in all studies. However it is envisaged that the methods are predictive in a statistically significant proportion of studies. It is to be understood that, if any prior art publication is referred to herein, such reference 25 does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country. In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, 30 i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. It must also be noted that, as used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. 4223871_1 (GHMatters) P89027.AU.1 25 It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification. 5 Brief Description of the Figures The invention will now be described with reference to the following non-limiting examples, in which: Figure 1 shows a schematic representation of HtrA3 protein domains and mAb epitope 10 locations. The domain structures of human HtrA3-L and HtrA3-S proteins are shown. The solid bars above or below the protein domains denote the epitope location of each mAb, the four mAbs on the top (6G6, 9C9, 1OH10 and 3E6) were raised against HtrA3-L-S305A protein, and the mAb on the bottom (2C4) was raised against a synthetic peptide. SP, signal peptide; IGFB, IGF-binding domain; Kazal, Kazal-type S protease inhibitor domain; trypsin, trypsin-like 15 protease domain; PDZ, PDZ domain. Figure 2 confirms HtrA3 mAb specificity. (A) An equal amount (50 ng) of recombinant (r) human HtrA proteins [rHtrA1 (Al), rHtrA2 (A2) and rHtrA3 (A3, HtrA3-L-S305A)] were separated on reducing 12% SDS-PAGE gels and analysed using Western blot with HtrA3 mAbs (6G6, 9C9, 2C4, 1OH10 and 3E6), anti-HtrAl and anti-HtrA2 antibodies respectively. (B) HtrA3-L 20 S305A (120 ng) was immunoprecipitated (IP) with each of the five HtrA3 mAbs and analysed using Western blot with the biotinylated mAb counterpart (WB mAb = biotinylated IP mAb). The control was the 2C4 IP products probed with a biotinylated irrelevant mAb. (C) Western blot analysis of 15 pl conditioned media of HEK-293F cells transfected with control plasmids (control transfection, C) or WT HtrA3-L (L) or HtrA3-S (S) constructs with the five HtrA3 mAbs. 25 Figure 3 shows Western blot analysis of protein lysates from human and mouse reproductive tissues using HtrA3 mAbs. Cell lysates (50 pg) and tissue lysates (25 pg) were separated on reducing 12% SDS-PAGE gels. p-actin served as a loading control. (A) Lysates from human trophoblast cell line HTR8 (HTR8) and human endometrial tissues of the mid proliferative (MP) and mid-secretory (MS) phases of the menstrual cycle were probed with 30 mAbs 6G6, 2C4, 1OH10 and 3E6. (B) Lysates from embryo implantation sites on d10.5 of pregnancy from wild-type (WT) or HtrA3 knockout (KO) mice were probed with mAbs 2C4 and 3E6. 4223871_1 (GHMatters) P89027.AU.1 26 Figure 4 shows immunohistochemical analysis of HtrA3 proteins in human and mouse reproductive tissues using HtrA3 mAbs. Representative images of immunostaining in human endometrial tissues from the mid-proliferative (MP, A and C) and mid-secretory (MS, B and D) phases of the menstrual cycle with mAbs 6G6 (A and B) and 1 OH 10 (C and D). Representative 5 images of immunostaining in mouse embryo implantation sites on d10.5 (E) and d13.5 (F) of pregnancy with mAb 3E6. Figure 5 is a schematic illustration of HtrA3 AlphaLISA principle. The AlphaLISA assay requires two HtrA3 antibodies recognising non-overlapping epitopes, one antibody is biotinylated and the other conjugated to acceptor beads. In the presence of HtrA3 proteins, the 10 streptavidin-coated donor beads (blue) are brought into close proximity to the anti-HtrA3-coated acceptor beads (red). When excited at 680nm, the Alpha Donor beads release singlet oxygen (02) which travels to the nearby Acceptor beads and induces a chemiluminescent emission at 615 nm, the AlphaLISA signal. Figure 6 illustrates the optimisation and validation of HtrA3 AlphaLISAs. Data is shown 15 for three independent AlphaLISAs employing different mAb pairs [denoted as conjugated (C)/biotinylated (B)]: 10H10C/6G6B, 10H10C/2C4B and 10H10C/3E6B. Assays were optimised using HtrA3-L-S305A as the standard with the 1 h (A-C) and overnight (D and E) protocols. (A) Detection of an equal amount (50 pM) of HtrA3, HtrA2, HtrA1 or BSA, or measuring HtrA3 (50 pM) when either a biotinylated irrelevant antibody (unrelated mAb) replaced the biotinylated 20 HtrA3 mAb or donor beads were absent. (B) Analysis of an equal amount of HtrA3 (50 pM) with various concentrations of biotinylated mAbs to determine the sub-hook point. (C) Serum matrix inhibition of the 10H10C/2C4B AlphaLISA. Serum was titrated with an equal amount (500 pM) of HtrA3. (D) Representative calibration curves of HtrA3-L-S305A in immuno buffer (IB) and serum using the overnight protocol. (E) Analysis of wild-type (WT) HtrA3 proteins with the 25 overnight AlphaLISA protocol. Conditioned media from HEK-293F cells transfected with WT HtrA3-L or HtrA3-S constructs were assayed, HtrA3 concentrations were determined from a calibration curve of HtrA3-L-S305A standard diluted in the transfection media. Figure 7 shows the detection of HtrA3 in serum from normal vs preeclamptic women using HtrA3 10H10C/2C4B AlphaLISA. (A) Previously published western blotting densitometric 30 analysis data of sera from 13-14 week pregnant women who subsequently developed preeclampsia (PE, n=8) or underwent normal pregnancies (Normal, n=8). Serum HtrA3 levels were significantly higher in preeclamptic women. (B) The same cohort of (A) except n=7 for the 4223871_1 (GHMatters) P89027.AU.1 27 PE group (one sample exhausted) was analysed using the HtrA3 10H10C/2C4B AIphaLISA with HtrA3-L-S305A standard diluted in serum Figure 8 shows real-time progressive curves of substrate hydrolysis by HtrA3 in the presence of 50ug/ml individual HtrA3 mAb (6G6, 9C9, 2C4, 10H10 and 3E6) or control mAb. 5 Figure 9 illustrates dose-dependent inhibition of HtrA3 activity by HtrA3 mAb 10H10. The data are expressed as reductions in the rate of substrate hydrolysis relative to the Control (control mAb at 50 ug/ml). ***, P<0.001. Figure 10 illustrates dose-dependent enhancement of HtrA3 activity by HtrA3 mAb 6G6. The data are expressed as increases in the rate of substrate hydrolysis relative to the Control 10 (control mAb at 50 ug/ml). ***, P<0.001. Figure 11 illustrates dose-dependent inhibition of HtrA3 activity following the addition of HtrA3 mAb 6G6. The data are expressed as reductions in the rate of substrate hydrolysis relative to the Control (HtrA3 mAb 6G6 at 50 ug/ml). ***, P<0.001. Figure 12 illustrates ELISA detection of wild-type recombinant HtrA3-L and HtrA3-S 15 isoforms in the media of COS-7 cells in which HtrA3-L or HtrA3-S was stably transfected. Control, control cells; L6, cells were transfected with HtrA3-L and S11 with HtrA3-S. (A) HtrA3 ELISA for the detection of total HtrA3, using HtrA3 mAb 10H10 as the capture antibody and HtrA3 mAb 9C9 as the detection antibody. (B) HtrA3 ELISA for the detection of HtrA3-L, using HtrA3 mAb 10H10 as the capture antibody and HtrA3 mAb 6G6 as the detection antibody. 20 Figure 13 illustrates ELISA detection of recombinant mammalian HtrA proteins HtrA1, 2, 3 and 4. All 4 HtrA proteins were at an equal concentration (90 pM). (A) HtrA3 ELISA for the detection of total HtrA3, using HtrA3 mAb 10H10 as the capture antibody and HtrA3 mAb 9C9 as the detection antibody. (B) HtrA3 ELISA for the detection of HtrA3-L, using HtrA3 mAb 10H10 as the capture antibody and HtrA3 mAb 6G6 as the detection antibody. In both 25 experiments, HtrA3 ELISAs detected HtrA3 only, and did not detect HtrA1, HtrA2 or HtrA4. Figure 14 illustrates representative standard curves of recombinant HtrA3 in standard diluent or serum diluent. Recombinant HtrA3-L-S305A was serially diluted in standard diluent or serum diluent. (A) HtrA3 ELISA for the detection of total HtrA3, using HtrA3 mAb 10H10 as the capture antibody and HtrA3 mAb 9C9 as the detection antibody. (B) HtrA3 ELISA for the 30 detection of HtrA3-L, using HtrA3 mAb 10H10 as the capture antibody and HtrA3 mAb 6G6 as the detection antibody. 4223871_1 (GHMatters) P89027.AU.1 28 Figure 15 illustrates ELISA detection of total HtrA3 in sera collected across gestation. HtrA3 ELISA for the detection of total HtrA3 (mAb 1OH10 as the capture antibody and mAb 9C9 as the detection antibody) was applied to 6 sera collected at 9 (wk9), 20 (wk20) and 34 (wk34) weeks of gestation from 2 pregnant women (3 samples across gestation per women). 5 Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques used by the inventor to function well in the practice 10 of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Abbreviations used: 15 AlphaLISA, Amplified luminescent proximity homogeneous assay-linked immunosorbent assay; DMEM, Dulbecco's modified Eagle's medium; ECI, enhanced chemiluminescent; ELISA, the enzyme linked immunosorbent assay; GST, glutathione S-transferase; HTR8, human trophoblast cell line; HtrA -1, -2, -3, high temperature requirement factor A -1, -2, -3; IB, immunoassay buffer; IGFB, insulin-like growth factor binding; IHC, immunohistochemistry; IP, 20 immunoprecipitation; IUGR, intrauterine growth restriction; KLH, Keyhole Limpet Hemocyanin; mAb, monoclonal antibody; MP, mid-proliferative; MS, mid-secretory; MSR, molecule saturation ratio; ON, overnight; PE, preeclampsia; QC, quality control; RIPA, radioimmunoprecipitation assay; TBS, Tris buffered saline, TBST; TBS containing 0.2% Tween20; WB, western blotting; WT, wild type. 25 Example 1: Materials and Methods: Recombinant human HtrA proteins C-terminally His-tagged full length HtrA1 protein (produced in insect cells) and mature 30 HtrA2 protein (produced in Esherichia coli) were purchased from Creative Biomart (Shirley, NY, USA). C-terminally His-tagged human wild type (WT) HtrA3-L produced in insect cells was 4223871_1 (GHMatters) P89027.AU.1 29 purchased from Protealmmun GmbH (Berlin, Germany). C-terminally His-tagged catalytically inactive HtrA3-L (HtrA3-L-S305A, where the catalytic site serine residue 305 was substituted with alanine) was synthesised using the wheat germ cell free technology as previously described and purified using Ni-NTA agarose (QIAGEN, GmbH, Hilden, Germany). 5 HtrA3 monoclonal antibody (mAb) production HtrA3 mAbs were produced at the antibody facility at The Walter and Eliza Hall Institute of Medical Research, Bundoora, Victoria, Australia, using standard protocols of the facility. In brief, BALB/c mice were injected intraperitoneally with 50 pg of HtrA3-L-S305A protein or 30 pg 10 of a synthetic peptide (TIKIHPKKKL, corresponding to aa 230-239 of both HtrA3-L and HtrA3-S, andconjugated to Keyhole Limpet Hemocyanin (KLH) through a C-terminal Cys; Mimotopes, Victoria, Australia). The mice received two additional injections of the same antigen/dose at the same site at 4 and 8 weeks later. Ten days after the third immunisation, the mice were bled and the sera was screened by ELISA on HtrA3-L-S305A protein or the peptide (without KLH 15 conjugation) to determine antibody titre. The sera were also screened using western blotting (WB) on HtrA3-L-S305A protein. The mice were rested for 4 weeks, and the highest respondent mouse in each immunisation group was selected for a final booster immunisation. Four days later, mice were sacrificed and blood and spleens collected. A single cell suspension of spleen cells was prepared, washed in serum-free Dulbecco's modified Eagle's medium (DMEM), and 20 fused with Sp2/0 myeloma cells in the presence of 50 % (w/v) polyethylene glycol 1500. The cells were resuspended in Hybridoma Serum Free Media (Invitrogen), supplemented with 10 % (v/v) FCS, HAT (hypoxanthine, aminopterin, and thymidine) media and IL-6. Cell suspensions were seeded in 96-well Falcon microwell flat-bottom plates (2 x 105 cells/well) and selected in HAT medium by overnight culture. After 10-12 days, the supernatants from hybridoma clones 25 were harvested and screened by ELISA on HtrA3-L-S305A protein or the peptide. The positive hybridomas were further confirmed on HtrA3-L-S305A protein using WB. Individual positive hybridomas were subcloned by standard limit dilution and screened using ELISA and then WB as specified above. HtrA3 mAbs were purified from clonal hybridoma supernatants using Protein A or G 30 sepharose columns, eluted into sterile PBS, isotyped and retested on HtrA3-L-S305A protein using WB. Purified mAbs were aliquoted and stored at -20'C for long term storage or at 4'C for frequent use. 4223871_1 (GHMatters) P89027.AU.1 30 Epitope mapping of mAbs The linear or continuous epitopes of the mAbs raised against HtrA3-L-S305A protein were determined by screening a custom-synthesised peptide library (PepSet, Mimotopes). The 5 full length amino acid sequence of human HtrA3-L (minus the signal peptide) was used to synthesise a complete set of 86 overlapping 15-aa peptides (overlap by 10 and offset by 5 aa). The N-terminus of each of the 86 peptides was conjugated to biotin followed by a 4-aa spacer SGSG and the C-terminus was amidated. The PepSet peptides were immobilised on streptavidin-coated/BSA-blocked 96-well plates (Pierce, Rockford, IL USA) and used for 10 screening hybridoma supernatants using standard ELISAs. The epitopes of the purified clonal mAbs were further confirmed by retesting them on the whole PepSet and on individual peptides of interest that were separately synthesised and purified (GL Biochem, Shanghai, China). Human endometrial tissues 15 Human endometrial tissue biopsies from the mid-proliferative (MP) and mid-late secretory (MS) phases of the menstrual cycle were obtained at curettage from women undergoing minor gynaecological surgical procedures, such as laparoscopic sterilisation or tubal patency. Written informed consent was obtained from all participants, and ethical approval was obtained from the Southern Health Human Research Ethics Committee, Melbourne, Australia. 20 Tissues were either snap-frozen in liquid nitrogen and stored at -80'C for protein extraction or fixed in buffered formalin (pH 7.4) and processed to paraffin wax blocks for immunohistochemistry (IHC) as previously described. Mouse uterine tissues 25 Uterine tissues from non-pregnant wild type (WT) mice (C57BL/6), and embryo implantation sites from pregnant day (d) 10.5 and d13.5 WT mice or d10.5 HtrA3 knockout (KO) mice (unpublished) were obtained as previously described. Tissues were immediately snap frozen in liquid nitrogen for protein extraction or fixed in formalin for IHC as for the human tissues. Mice were housed and handled according to Monash University Animal Ethics 30 guidelines on the care and use of laboratory animals. All studies were approved by the Animal Ethics Committee at the Monash Medical Centre, Melbourne, Australia. 4223871_1 (GHMatters) P89027.AU.1 31 Cell culture and transfections The trophoblast HTR-8/SVneo (HTR8) cell line, kindly provided by Dr Charles Graham (Queen's University, Kingston, ON, Canada), was cultured as previously described. When confluent, cells were trypsinised, pelleted and stored at -80'C until protein extraction. 5 HEK-293F cells (Life Technologies, Mulgrave, VIC, Australia) were cultured in DMEM media (Life Technologies) with 10 % FCS. For transient transfection, HEK-293F cells were seeded (8 x 105 cells/well) in 6-well plates (Nunc) precoated with poly-D-lysine (Sigma) and cultured overnight at 37 0 C in 5 % CO 2 . The following day, cells were transfected with 5 pg of HtrA3 expression constructs [full length human HtrA3-L or HtrA3-S in pcDNA-DEST40 vector 10 (Invitrogen) with a C-terminal His-tag] or pcDNA empty vector (control transfection) using Lipofectamine 2000 and OPTI-MEM media (both from Invitrogen) according to the manufacturer's protocol. Cells were further cultured for 48 hours at 37 0 C before collection of media for analysis. 15 Protein lysate preparation from tissues and cells Frozen tissues were thawed on ice and lysed into radioimmunoprecipitation assay (RIPA) buffer (65 mM Tris, 1% NP40, 0.25% sodium deoxycholate, 154mM NaCl, 1 mM EDTA pH 7.4) containing protease inhibitors (Roche, Castle Hill, NSW, Australia) by freeze/thawing three times (using liquid nitrogen and a 37 0 C water bath) and grinding using ReadyPrep 20 Grinders (Biorad, Hercules, CA, USA) until the mixture was homogenous. DNA was sheared by passing the lysate through a 21 g needle at least 15 times using a 1 mL syringe, debris was removed by centrifugation (17, 000 g/10 min at 4 0 C) and the supernatant collected, aliquoted and stored at -20 0 C until use. Cell pellets were thawed on ice, lysed by resuspending in RIPA buffer containing 25 protease inhibitors, passed through a 21 g needle at least 15 times with a 1 mL syringe. After incubation on ice for 10 minutes, the cell debris was removed by centrifugation (10, 000 g/5 min at 4 0 C) and the lysate was aliquoted and stored at -20 0 C until use. Western blotting (WB) 30 Recombinant HtrA1, HtrA2 and HtrA3-L-S305A protein (50 ng), HTR8 cell lysates (50 pg), tissue protein lysates (25 pg), or neat conditioned media (15 pl) from HEK-293F cells transfected with control, WT HtrA3-L or HtrA3-S constructs, were analysed using standard WB (12% reducing SDS-PAGE and PVDF membrane). Protein concentrations were determined 4223871_1 (GHMatters) P89027.AU.1 32 using BCA assay kits (Pierce). Primary antibodies, including HtrA3 mAb hybridoma supernatants (1:1 dilution), purified clonal HtrA3 mAbs (50 pg/mL final concentration), anti HtrA1 (sc-1 5465) and anti-HtrA2 (sc-1 5467) antibodies (400 ng/mL final concentration, both are affinity-purified goat polyclonal, Santa Cruz Biotechnology, Santa Cruz, CA, USA), were 5 incubated overnight at 4'C and probed with secondary conjugates, rabbit anti-mouse IgG-HRP (1:5000, Cell Signalling, Beverly, MA, USA) or rabbit anti-goat IgG-HRP (1:4000, DAKO, Carpinteria, CA, USA) for 1 h at room temperature. Proteins were visualised with Pierce ECI Western Blotting Substrate (Thermo Fisher Scientific, Rockford, IL, USA) and Amersham Hyperfilm ECI film (GE Healthcare, Buckinghamshire, UK). To confirm protein loading, 10 membranes were stripped with Reblot Plus Strong (Millipore, Temecula, CA, USA) according to manufacturer's instructions and probed with anti-p-actin-HRP antibody (1:2000, Cell Signalling). Immunoprecipitation (IP) Purified clonal HtrA3 mAb (5 pg) was incubated with washed protein G agarose beads 15 (Roche) in RIPA buffer at 4'C for ~ 3 h on a rotating wheel. The HtrA3 mAb-bound beads were pelleted (235 g/40 sec), washed 3 times with ice cold RIPA buffer, and resuspended in RIPA buffer containing protease inhibitors (Roche). HtrA3-L-S305A (125 ng/IP) was added to the mAb-bound bead suspension, incubated at 4'C overnight on a rotating wheel and pelleted (3200 g/30 sec). After the pellet was washed 3 times with ice-cold RIPA buffer and centrifuged 20 (3200 g/30 sec), a 1:3 ratio of 4 x reducing SDS sample buffer (200 mM Tris-HCI pH 6.8, 4% w/v SDS, 40% v/v glycerol, 0.08% w/v bromophenol blue) containing 5% P-mercaptoethanol and immunoprecipitated HtrA3 was analysed using WB (25 pL) with the biotinylated HtrA3 mAb counterpart (WB mAb = IP mAb with biotinylation) and streptavidin-HRP conjugate (1:1500, ECI protein biotinylation kit; GE Healthcare Bioscience, Uppsala, Sweden). The mAbs were 25 biotinylated using the ECI protein biotinylation kit (GE Healthcare) according to manufacturer's instructions. Briefly, 100 pg mAb was incubated with 4 pL biotinylation reagent in bicarbonate buffer in a total volume of 200 pL for 1 h at room temperature on a rotating wheel. After the removal of free biotin with Zeba Spin Desalting Columns (Thermo Scientific) without buffer exchange, the sample was stored at 4'C with 0.1% sodium azide. 30 Immunohistochemistry (IHC) Tissue sections (5 pm) were subjected to standard IHC, using HtrA3 mAbs or non immune mouse IgG (Dako) as primary antibodies (8 pg/ml final concentration) and biotinylated 4223871_1 (GHMatters) P89027.AU.1 33 horse anti-mouse IgG antibody (1:200, Vector laboratories, Burlingame, CA, USA) as the secondary antibody in conjunction with Vectastain@ Elite ABC kit (Vector laboratories). Antigen was retrieved by microwaving the sections for sequential 2 x 5 min in 10 mM citrate buffer (pH 6.0) for human sections or in 1 mM EDTA (pH 8.0, containing 0.05 % Tween 20) for mouse 5 sections. Positive immunostaining (brown precipitate) was revealed by the application of the peroxidase substrate 3,3'-diaminobenzide (DAB) using liquid DAB Chromogen System (Dako). All sections were counterstained with Harris hematoxylin and analysed under an Olympus BH2 microscope fitted with a Fujix HC-2000 high-resolution digital camera (Fujix, Tokyo, Japan). 10 Serum from pregnant women Maternal serum samples from 13-14 week pregnant women as published were used. The preeclampsia group (n=7) developed preeclampsia later in their pregnancy. The controls (n=8) were matched for maternal age, gestation, parity and smoking status. Serum samples were also collected from two normal pregnant women at three time points during pregnancy (9, 15 20 and 34 weeks of gestation). Ethical approval was obtained from Southern Health Human Research Ethics Committee. Development and validation of HtrA3 AlphaLISAs Conjugation of mAbs to acceptor beads 20 Acceptor beads (Perkin Elmer, Waltham, MA, USA) were conjugated according to manufacturer's instructions. Briefly, for small scale testing, 10 pg mAb and 0.1 mg acceptor beads (10:1 ratio) were mixed with 130 mM phosphate buffer (pH 8.0) containing 0.06 % Tween-20 in a final volume of 20 pL and conjugated with the addition of freshly prepared NaBH 3 CN (final concentration 1.25 mg/mL). For large scale preparations, 100 pg mAb was 25 mixed with 1 mg acceptor beads in a final volume of 200 pL. The mixture was incubated for 48 h at 37'C on an orbital shaker, the unreacted sites were blocked with 3.25 mg/mL carboxymethoxylamine (CMO) for 1 h, and the conjugated beads were pelleted in a microfuge (17,000 g/15 min) at 4'C. The beads were washed twice with cold Tris buffer (100 mM, pH 8.0), pelleted (17,000 g/15 min) at 4'C, resuspended to 5 mg/mL in ice-cold PBS buffer containing 30 0.05 % Proclin-300 (storage buffer), vortexed and sonicated by pulsing for 20 sec with 1 sec intervals at 30 % amplitude (Vibracell; Danbury CT, USA). Conjugated beads were stored at 4'C in a screw capped tube. 4223871_1 (GHMatters) P89027.AU.1 34 Biotinylation of HtrA3 mAbs HtrA3 mAbs were biotinylated according to manufacturer's instructions. Briefly, 100 pg mAb was incubated with 93.7 pM Chromalink Biotin 354S (Solulink, San Diego, CA, USA,) in 200 pl PBS (pH 7.4) for 2 h at room temperature on a rotating wheel. Free biotin was removed 5 with Zeba Spin Desalting Columns (Thermo Scientific) with PBS buffer exchange. The concentration of biotinylated antibody, molecule substitution ratio (MSR; molecules of biotin per antibody) and antibody recovery were determined by the absorbance at 280, 354 and 450nm with PBS as the blank. After the adjustment of the final concentration to 500 nM with PBS/0.1 % Tween-20 and 0.05 % Proclin-300, the biotinylated antibody was dispensed into 25 pL aliquots 10 in screw capped tubes and stored at 4 0 C. Human serum matrix free of HtrA3 To deplete endogenous HtrA3 in serum, 10 pL streptavidin sepharose beads (Cell Signalling) per mL of serum (pool of 3 individual samples, free of known infection, obtained from 15 The Australian Red Cross) were washed once in 5 volumes PBS by vortexing and centrifugation (16,000 g/5 min) in a microcentrifuge. The beads were mixed with biotinylated HtrA3 10H10 (10H10B) mAb (625 fmole biotinylated mAb /pL beads) in 20 volumes of PBS/beads on a rotating wheel at room temperature for 2 h, pelleted (16,000 g/5 min) in a microfuge and washed once with 5 volumes of PBS/beads. The 10H10B-bound beads were resuspended in 20 the serum to be depleted and incubated at 4 0 C overnight on a rotating wheel. The mixture was centrifuged (16,000 g/5 min) in a microfuge and the HtrA3-depleted serum was dispensed into 150 pL aliquots and stored at -20 0 C. AlphaLISA 25 Alphalisa assays were performed in 384 well microplates (Alphaplates, Perkin Elmer) with each sample in triplicates according to manufacturer's instructions. HtrA3-L-S305A was used as the HtrA3 standard throughout. Briefly, 5 pL of analyte (sample or standard) was first added to wells, 10 pl of 10 nM biotinylated mAb (2 nM final) and 10 pL of 50 pg/mL mAb conjugated AlphaLISA Acceptor beads (10 pg/mL final) were then sequentially added and the 30 plates sealed with Top Seal (Perkin Elmer). For the 1 h standard protocol, the plates were incubated at room temperature (22-23 0 C) for 1 h on an orbital plate shaker (300 rpm). For the overnight protocol, the incubation was at 4 0 C overnight (16-19 hours). After incubation, 25 pL streptavidin-Donor beads solution at 80pg/mL (prepared in the dark) was added to each well 4223871_1 (GHMatters) P89027.AU.1 35 (40 pg/mL final) and incubated in the dark for 30 min at room temperature on a plate shaker (300 rpm). The europium luminescence was recorded on an Alphascreen compatible reader (Wallac EnVision Multilabel Plate reader equipped with the AlphaScreen module; Perkin Elmer). Data was imported into Masterplex Readerfit software (Hitachi Solutions America, South San 5 Francisco, CA, USA). The HtrA3 concentrations of unknowns were determined from a standard calibration curve fitted using four parameter logistic (4PL) non-linear regression from the above background counts of triplicate wells. The assay was first optimised using HtrA3-L-S305A as the standards diluted (1000, 500, 50, 10, 5, 1, 0.5, 0.05 and 0 pM) in Immunoassay buffer (IB) (Perkin Elmer). To select the best 10 antibody combinations, all five HtrA3 mAbs were separately conjugated or biotinylated on a small scale and all possible combinations (one biotinylated and one conjugated, a total of 5 x 4 = 20 combinations) were tested on a constant 50 pM HtrA3 standard. The best combinations (highest signal to background ratio) were selected for further optimisation. The antibody combinations were tested in three different assay buffers (Immunoassay, Universal 15 and HiBlock buffers, all from Perkin Elmer), to determine optimum signal to background ratios. The optimal biotinylated antibody concentration was determined by assaying various concentrations (10, 5, 4, 3, 2, 1 and 0.5 nM final) of the biotinylated antibody on a constant 50 pM HtrA3 standard. The average above-background counts of triplicates were plotted against the biotinylated antibody concentration and the peak (hook) point on the curve was 20 identified; the optimal antibody concentration was determined as the pre-hook (sub-hook) point concentration. The assay was further optimised by lengthening the primary incubation from 1 h at room temperature to overnight at 4'C. The assays were then optimised to detect HtrA3 in human serum by diluting HtrA3 standards in HtrA3-depleted human serum. The optimised protocols were tested for assay accuracy, reliability and repeatability. 25 Quality control (QC) samples were prepared by spiking purified insect-cell expressed WT HtrA3 L in IB or HtrA3 depleted serum. For each assay, 5 pL of HtrA3 standards or QC samples diluted in buffer or HtrA3-depleted serum were assayed with the overnight protocol. The percentage recovery (accuracy) of the standards in the calibration curve was determined by comparing the back-calculated concentration of standards to the nominal input concentration. 30 The intra-assay variation (reliability) for both the calibration curve and QCs was calculated from one assay of 10 replicates and the inter-assay variation (repeatability) from five (for calibration curve) or three (for QCs) independent assays performed at least in triplicate on separate days. The limit of detection (LOD) for each assay in each matrix was determined from 4223871_1 (GHMatters) P89027.AU.1 36 the mean of zero concentrations plus three times the standard deviation (SD) from the five assays used for the calibration curves. When the zero concentration was beyond the parameters of the 4PL equation, the lowest calculated value in the calibration curve was used. The lowest limit of quantification (LLOQ) was calculated as three times the LOD. 5 To detect HtrA3 in conditioned cell media, 5 pL HtrA3 standards diluted in the transfection media were assayed together with 5 pL of conditioned media from cells transfected with HtrA3-L or HtrA3-S constructs using the overnight protocol. To detect HtrA3 in pregnant human serum, 5 pL HtrA3 standards prepared in HtrA3-depleted serum were assayed together with 5 pL human serum samples using the overnight protocol. Original concentrations of HtrA3 10 in the unknown samples were calculated by multiplying the concentration calculated from the calibration curve by the assay dilution factor (x10). Unpaired two-tailed t-test was performed (Prism v 5.03, GraphPad Software Inc., San Diego, CA, USA) for statistical analysis of PE versus control samples, with P<0.05 (*) being significant and P<0.01 (**) highly significant. The correlation (rho [r] with P value) between western blot data and AlphaLISA data was assessed 15 with Spearman's rank correlation test. RESULTS Generation of HtrA3 mAbs HtrA3-L and HtrA3-S contain identical domains, except HtrA3-S lacks the C-terminal 20 PDZ domain (Figure 1). Wild type HtrA3 proteins, like other proteases, are susceptible to auto cleavage, difficult to purify in a large amount and unstable for long-term storage. Substituting the serine with alanine (S305A) in the catalytic site abolishes protease activity and stabilises HtrA3. This single substitution mutant (HtrA3-L-S305A) with a GST tag has previously been readily synthesised using a wheat-germ cell-free translation system, HtrA3-L-S305A with a His 25 tag was synthesised and purified in this study. To produce HtrA3 mAbs, mice were immunised against HtrA3-L-S305A or a synthetic peptide corresponding to aa 230-239 of both HtrA3-L and HtrA3-S. This led to the cloning and purification of five distinct HtrA3 mAbs: 6G6-17-10-2 (6G6), 9C9-9-10 (9C9), 2C4-1-8 (2C4), 1OH10-22-29-6 (1OH10) and 3E6-3-32 (3E6) respectively (Figure 1). Four (6G6, 9C9, 1OH10 30 and 3E6) of these were from immunisation against HtrA3-L-S305A and one (2C4) against the peptide. 6G6 was isotyped as IgG2b and the other four IgG1. The linear epitope(s) of the four HtrA3-protein-derived mAbs were mapped to distinctive regions on the protein (Figure 1). 4223871_1 (GHMatters) P89027.AU.1 37 Confirmation of HtrA3 mAb specificity on recombinant human HtrA proteins The HtrA3 mAbs were tested using WB on recombinant human HtrA family members HtrAl -3, where HtrA3-L-S305A was used as HtrA3 (Figure 2A). All mAbs detected HtrA3 only and not HtrAl or HtrA2, confirming their high specificity to HtrA3. The integrity of rHtrA1 and 5 rHtrA2 was validated using anti-HtrAl and anti HtrA2 antibodies respectively (Figure 2A). All mAbs also recognised HtrA3-L-S305A using immunoprecipitation (Figure 2B). To confirm that the mAbs recognise wild-type (W)T HtrA3, HEK-293F cells were transiently transfected with HtrA3-L or HtrA3-S constructs, or control plasmids, and their media were analysed using WB (Figure 2C). While 9C9, 2C4 and 1OH10 detected both HtrA3-L and 10 HtrA3-S, 6G6 and 3E6 recognised HtrA3-L only, consistent with their epitope(s) containing HtrA3-L-specific sequences (Figure 1). The major bands for HtrA3-L (- 50 kDa) and HtrA3-S (~ 41 kDa) are consistent with their predicted sizes, the smaller minor bands likely represent HtrA3 cleavage products. With the exception of 9C9 detecting a non-specific -18 kDa band, the other four mAbs were highly specific to HtrA3 as no bands were seen in control transfection. 15 This confirmed that all HtrA3 antibodies, either against HtrA3-L-S305A or the peptide, recognise WT human HtrA3 proteins in an isoform-specific manner. Application of HtrA3 mAbs in western blot analysis of human and mouse tissues The five HtrA3 mAbs were next tested using WB on endogenous HtrA3 proteins in 20 human or mouse tissues. For human samples, protein lysates from placental cell line HTR8 and human endometrial tissues of the mid-proliferative (MP) and mid-late secretory (MS) phases of the menstrual were analysed. While 9C9 did not recognise HtrA3 specifically (data not shown), the other four mAbs (6G6, 2C4, 1OH10 and 3E6) detected different isoforms/cleaved forms of HtrA3 proteins (Figure 3A) with higher levels in the MS compared to MP phase, consistent with 25 previous findings. As mouse and human HtrA3 are 95% similar, the mAbs were also tested on mouse uterine tissue lysates. Of the five mAbs, only 2C4 and 3E6 recognised a major specific band corresponding to HtrA3-L in implantation site lysate (Figure 3B), consistent with HtrA3-L being predominantly expressed in mice. 30 These results demonstrate that the five HtrA3 mAbs recognise HtrA3 proteins in the human versus mouse with different specificities. 4223871_1 (GHMatters) P89027.AU.1 38 Application of HtrA3 mAbs in IHC on human and mouse tissues All five mAbs were tested using IHC on formalin-fixed human and mouse uterine tissues. While 1OH10 and 6G6 specifically stained the luminal and glandular epithelium in human endometrial tissues with a higher level in the MS than MP phase (Figure 4 A-D), only 3E6 5 specifically stained HtrA3 in the mouse implantation sites with a higher level on d10.5 than d13.5 (Figure 4 E-F), consistent with previously observed patterns using a HtrA3 specific sheep antibody. These results further demonstrate that the five HtrA3 mAbs have different specificities towards human versus mouse HtrA3 proteins. 10 Development of high throughput, specific and sensitive HtrA3 AlphaLISA assays Selection of the best mAb pairs for HtrA3 AlphaLISA To measure HtrA3 levels in biological fluids, the HtrA3 mAbs were incorporated into an AlphaLISA (Figure 5). As AlphaLISA requires two distinct antibodies, one conjugated to acceptor beads and the other biotinylated, all five mAbs were separately conjugated or 15 biotinylated and all possible 20 (5 x 4) combinations tested in immunoassay buffer (IB). The best combinations were when 1OH10 was conjugated to the acceptor beads (10H10C) and paired with any of the other four biotinylated mAbs, with a signal to background ratio 6-30 times higher than any other combinations (data not shown). When 1OH1OC was paired with a biotinylated mAb (xB), the best pairings were in the order of 6G6B>2C4B>3E6B>9C9B (data 20 not shown). The best three pairs, 1OH10C/6G6B, 1OH10C/2C4B and 1OH10C/3E6B, were further tested. HtrA3 AlphaLISA specificity All three HtrA3 ALphaLISAs (1OH10C/6G6B, 1OH10C/2C4B and 1OH10C/3E6B) were 25 specific to HtrA3 and did not detect HtrAl, HtrA2 or BSA (Figure 6A). HtrA3 was not detected when the donor beads were omitted or when an unrelated biotinylated mAb was substituted for the HtrA3 biotinylated mAb (Figure 6A). Optimisation of HtrA3 AlphaLISA assay for serum detection 30 To determine the optimal concentration of the biotinylated antibody in the AlphaLISAs, 50 pM of HtrA3 standard was assayed in IB with various concentrations of biotinylated antibodies (Figure 6B). All three antibody pairs responded in a similar fashion and the sub-hook point was 2 nM for three assays. 4223871_1 (GHMatters) P89027.AU.1 39 We next tested the matrix effect of serum in the assay. Spiking 5 pL neat serum (representing 10% of the total assay volume) in the 10H10C/2C4B AlphaLISA with 500 pM HtrA3 standard decreased the signal 7.6 fold. In general, the signal was inversely proportional to the amount of serum in the assay (Figure 6C). This highlighted the need to construct HtrA3 5 standard curves in HtrA3-depleted serum when examining serum. The protocol thus far optimised involved the incubation of the analyte, 10H1OC acceptor beads and biotinylated antibodies in IB for 1 h, before the addition of the straptavidin-donor beads. To increase the signal, the 1 h incubation (1 h protocol) was extended to overnight (ON protocol) which doubled the signal (data not shown). The ON protocol was thus used for further 10 studies. HtrA3 calibration curves To account for the serum matrix effect, the HtrA3 standard (HtrA3-L-S305A) was diluted in HtrA3-depleted serum or IB respectively, and calibration curves in IB and serum for all three 15 antibody pairs were constructed using optimised conditions (Figure 6D). The curves were linear between 1 - 1000 pM for all three assays in both matrices (Figure 6D). For each HtrA3 concentration and for all three mAb pairs, the signal was 5-11 fold lower in serum than in IB (Figure 6D). 20 Testing HtrA3 AlphaLISAs on WT HtrA3 proteins We next tested whether the three AlphaLISAs could recognise WT HtrA3 in the media of transfected cells. The 10H10C(Conjugated)/2C4B(Biotinylated) AlphaLISA detected both HtrA3 L and HtrA3-S, whereas the 10H10C/6G6B and 10H10C/3E6B AlphaLISAs recognised HtrA3-L only (Figure 6E), consistent with 6G6 and 3E6 being HtrA3-L specific (Figure 2C). Both 25 10H10C/2C4B and 10H10C/6G6B detected similar concentrations of HtrA3-L, however, the 10H10C/3E6B AlphaLISA detected a lower concentration (Figure 6E). Accordingly, the 10H10C/6G6B AlphaLISA was selected to detect the HtrA3-L isoform and the 10H10C/2C4B AlphaLISA to detect both HtrA3 isoforms. 30 HtrA3 AlphaLISA working range and intra- and inter-assay variations The accuracy and precision of the 1 OH 1 0C/6G6B and 1 OH 1 0C/2C4B AlphaLISAs were further determined using calibration curves in IB or serum (Table 1). The average recovery percentages between 5 - 1000 pM were highly accurate as the back calculated concentrations 4223871_1 (GHMatters) P89027.AU.1 40 were within 3% of the nominal concentrations. The calibration curve was also precise with average intra-assay variations of 7 - 11 %. Overall, assays in serum matrix showed higher variability than in IB, and the 10H10C/6G6CB assay had higher variability than 10H10C/2C4B for both IB and serum. The estimation of concentration was also very reproducible within this 5 range as reflected by the low inter-assay variation (1 - 4%). The intra-assay variation was higher than the inter-assay variation. When the lowest limit of quantification was calculated (Table 2), the lower limit of 5 pM was acceptable for all except 10H10C/6G6B in serum which has a calculated LLOQ at 24.03 pM. Thus, in serum, the working ranges are 24 -1000 pM (1176 49000 pg/mL) for 10H10C/6G6B and 5 - 1000 pM (245 - 49000 pg/mL) for 10H10C/2C4B. The 10 upper limit of quantitation was not determined. The two assays were further tested on active recombinant HtrA3 spiked in IB or serum as QC samples. The assays were reliable with intra-assay variation below 10%, and repeatable with most inter-assay variations under 14% (Table 3). 15 Application of HtrA3 AlphaLISA to detect preeclampsia in early pregnancy The validity of the 10H10C/2C4B AlphaLISA was further confirmed by testing a previously published cohort of sera from pregnant women at 13-14 weeks of gestation. Using western blotting, serum HtrA3 levels at this gestation were found to be significantly higher in women who subsequently developed PE (PE, n=8) than those who had normal pregnancies 20 (Normal, n=8) (replotted in Figure 7A). The AlphaLISA detected a similar difference (P<0.04) between the PE and Normals (Figure 7B). Spearman's rank correlation analysis showed a significantly positive correlation between the western blot and the AlphaLISA data (r = 0.539, P<0.038). The various applications of the five HtrA3 mAbs in mouse and human are summarised in 25 Table 4. Table 1 Accuracy and precision of 10H10C/2C4B and 10H10C/3E6B AlphaLISAs on HtrA3 standard Concentrations of HtrA3 (HtrA3-L-S305A) were back calculated from the HtrA3 calibration 30 curve and the inter- and intra-assay variations determined using AlphaLISAs in IB and analyte depleted human serum matrix respectively. Results are from five independent experiments; three assays in triplicate and two assays in ten replicate wells. The intra-assay variation was calculated from one assay of ten replicate wells. 4223871_1 (GHMatters) P89027.AU.1 cu

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0 -- t ( 'N m uC ~ c ) Z',C : C') o ~ ( C)04C: CD 0 U) > L 000 a) ) ~ 0) 0 000:C'It 0 ) r- C%4 T7 qq- m- CS0 :r x-) qC :C a) C) 00 CU 0 co 04 a "t0 c)0 C (1) ) )(N 0 )C\ C (C) CD )N 0 0 U) r- (0cI )0 > U-) c) o r )O N-0-0 0 m O C4C)CO C1:0 (0 0) ~ 00:C < ) r- m c LO:~ 00L Co- 0 r, 000 o )U) . (0 0NC ZO a C=a) ~~~)> a)0 Y)0:0 Qa)C4 9 o 42 Table 2 Limit of detection and lowest limit of quantitation of the HtrA3 AlphaLISAs The limit of detection (LOD) of 10H10C/6G6B and 10H10C/2C4B AlphaLISAs was determined for two matrices (lB and analyte-depleted human serum) from the mean of zero concentration plus three standard deviations in the five assays from Table 1. The lowest limit of 5 quantitation (LLOQ) is three times the LOD. Antibody pair in AlphaLISA Matrix LOD (pM) LLOQ (pM) IB 1.77 5.32 10H10C/6G6B Serum 8.01 24.03 IB 0.97 2.90 1OH1 0C/2C4B Serum 1.81 5.44 Table 3 Reliability and repeatability of the 10H1OC/2C4B and 10H1OC/3E6B AlphaLISAs on quality control samples 10 The inter- and intra-assay variations of the two HtrA3 AlphaLISAs detecting QCs in IB and serum were determined using a HtrA3-L-S305A calibration curve in IB or analyte-depleted human serum. QCs representing high and medium concentrations of HtrA3 were created in bulk by spiking purified insect-expressed WT HtrA3-L protein in buffer and analyte-depleted human serum matrix respectively and frozen in aliquots. Results are from three independent 15 experiments; two assays in triplicate and one assay in ten replicate wells. The intra-assay variation was calculated from one assay of ten replicate wells. AlphaLISA mAb pairs: Conjugated (C)/Biotinylated (B) 10H10C/6G6B 1 OH10C/2C4B Amount of HtrA3 I ntra-assay Inter-assay Intra-assay % CV Inter-assay %CV % CV %CV ___________ In buffer High - 390 pM 9.80 14.70 8.80 10.61 Medium - 50 pM 5.81 10.01 6.73 11.22 Average CoV % 7.81 12.35 7.77 10.92 In serum High - 590 pM 8.76 19.82 9.62 16.43 Medium - 75 pM 5.76 16.15 8.97 10.90 Average CoV % 7.26 17.98 9.29 13.66 42238711 (GHMatters) P89027.AU.1 43 Table 4 Summary of HtrA3 mAb applications HtrA3 mAb Application Protein source 6G6 9C9 2C4 10H10 3E6 rHtrA3 Western blot Human tissues Mouse tissues Immuno precipitation rHtrA3 Immuno- Human tissues histochemistry Mouse tissues HtrA3-L+S 1OH10C/ 1OH10C/2C4B AlphaLISA* 2C4B HtrA3-L 1OH10C/6G6B 1OH10C/6G6B ELISA HtrA3-L+S Detection Capture HtrA3-L Detection Capture *For AlphaLISA, the mAb pairing and modifications [either conjugated (C) or biotinylated (B)] are shown 5 4223871_1 (GHMatters) P89027.AU.1 44 Discussion The inventors generated and cloned five HtrA3 mAbs (6G6, 9C9, 2C4, 1OH10 and 3E6) with unique epitopes, three (9C9, 2C4 and 10H10) of which recognise both HtrA3-L and HtrA3 5 S isoforms and two (6G6 and 3E6) detect HtrA3-L only. These mAbs are highly specific to HtrA3 and do not recognise human HtrA1 or HtrA2. The antibodies are confirmed to be applicable in western blotting and IHC analysis of endogenous HtrA3 proteins in the mouse and human tissues. We have further established and validated two HtrA3 AlphaLISA assays using two different antibody pairs, one detecting both HtrA3-L and HtrA3-S isoforms (1OH10C/2C4B) 10 and one detecting the HtrA3-L isoform only (10H10C/6G6B). These assays are highly specific to HtrA3 and suitable for large scale screening of human serum with a lower limit of quantitation of 5 - 24 pM and 7 - 18% intra- and inter-assay variations. The 1OH10C/2C4B AlphaLISA .n Protein HtrA3 mAb ApplicationPrti _____________________________ source 6G6 9C9 2C4 1OH10 3E6 rHtrA3 Human Western blot tissues Mouse tissues Immunoprecipitation rHtrA3 - Human Immunohistochemistry uses tissues HtrA3-L+S 10H10C/2C4B IOH10C/2C4B AlphaLISA* HtrA3-L 1OH10C/6G6B 1OH10C/6G6B recognising both HtrA3 isoforms detects significantly higher levels of HtrA3 in serum at early gestation in pregnant women who subsequently developed PE than controls as previously 15 published. These HtrA3 mAbs provide critical tools for the diagnosis of PE and possibly other human diseases associated with HtrA3 dysregulation, and will enable further characterisation of HtrA3 isoform-specific biology. The five HtrA3 mAbs were first validated using WB for specificity using recombinant HtrA3 proteins, and then evaluated on endogenous HtrA3 in human and mouse tissues using 20 WB and IHC. In WB analysis, four mAbs (6G6, 2C4, 1OH10 and 3E6) detected endogenous 4223871_1 (GHMatters) P89027.AU.1 45 proteins in human tissues and two (2C4 and 3E6) in mouse tissues; the band patterns were similar to that of previously published sheep polyclonal HtrA3 antibodies In IHC, two mAbs (6G6 and 1OH10) specifically immunostained HtrA3 in human endometrial tissues and one (3E6) recognised HtrA3 in mouse tissues; the staining was highly similar to IHC using the sheep 5 antibodies. Thus mouse and human specific antibodies for different applications were identified. This in turn highlighted that not all five HtrA3 mAbs were suitable in all applications and for all species, suggesting that human and mouse HtrA3 proteins differ in their epitope formation despite high sequence similarities. Three of the HtrA3 mAbs were used to develop AlphaLISAs that are robust, highly 10 sensitive and suitable for high throughput screening of HtrA3 isoforms in serum at picomolar levels across at least three orders of magnitude. In AlphaLISA, the antibody pair 10H10C/2C4B detected both HtrA3-L and HtrA3-S isoforms, whereas 10H10C/6G6B recognised HtrA3-L only. Importantly, these HtrA3 AlphaLISA assays only required a small volume (5 pL) of serum and were optimal for analysing undiluted serum with LLOQs and intra- and inter-assay variations 15 comparable to other homogenous assays and Alphascreens. Notably, the assay for both HtrA3 isoforms detected significantly higher levels of HtrA3 at 13-14 weeks of gestation in pregnant women who subsequently developed PE later in gestation, compared with women who had a normal pregnancy. The concentrations detected in the original human pregnant serum ranged from 118 - 308 pM (5782 - 15 092 pg/mL) in normotensive women and 135 to 765 pM (6615 20 37 485 pg/mL) in women who developed PE. However, as the samples had been used in a previous experiment (thawed multiple times) they were sub-optimal. Additional studies reported in Example 3 utilise HtrA3 ELISAs to screen serum samples to establish the robustness of HtrA3 as an early PE marker. Earlier detection of PE would greatly facilitate careful monitoring and supportive 25 therapies to reduce morbidity and mortalities in both the mother and baby. Currently there are no clinical assays with a high enough positive predictive value for early detection of PE. Financially, advanced notice prior to the development of PE would reduce the overall costs involved in healthcare by strategic focussing on high risk women rather than all pregnant women 30 The causes of PE may be multi-factorial and thus combining a number of markers would be beneficial to diagnose PE. There is the potential of combining one or several markers such as cell free DNA/RNA, placental protein 13 (PP13), pentraxin 3, pregnancy associated plasma 4223871_1 (GHMatters) P89027.AU.1 46 protein A, and placental growth factor with the biophysical marker Doppler uterine artery ultrasound and predisposing maternal risk factors. The advantage of using HtrA3 as an early marker for PE over the other potential early markers such as PP1 3 is that HtrA3 is upregulated rather than downregulated. It is possible that combining HtrA3 detection with other markers may 5 strengthen the power of PE detection. In summary, HtrA3 mAbs developed in this study will enable thorough investigations into the mechanisms of isoform-specific actions of HtrA3 in ovary development, placental development and cancer progression, and provide a means to characterise the effects of dysregulation of HtrA3 in these processes. These mAbs will also broaden the development of 10 other immunoassays where dysregulation of HtrA3 is a disease marker. The newly developed HtrA3 AlphaLISA assays are suitable for large scale screening of human fluids and have been validated on human serum. Example 2: 15 Identification and confirmation of inhibitory and stimulatory HtrA3 mAbs Methods - HtrA3 activity assay Purified C-terminally His-tagged WT human recombinant human HtrA3-L produced in insect cells was purchased from Protealmmun GmbH (Berlin, Germany). Protease activity of HtrA3-L was determined by the cleavage of a custom-made p-nitroaniline labelled peptide 20 substrate (Ac-VFNTLPMMGKASPV-pNA, synthesised by GL Biochem Ltd., Shanghai China) known to be specific to mammalian HtrA proteolysis (1). Protease activity of HtrA3-L in the presence or absence of HtrA3 mAbs was determined in a 50 pL reaction in half-area 96-well clear-bottom black plates (Sigma). First, a 46.5 pL reaction mixture containing 5 pL of rHtrA3-L (final concentration 0.2 pM) or buffer control, 2.5 pL HtrA3 mAb (final concentration 50 pg/ml) or 25 control mAb (a non-HtrA3 mAb produced/purified similarly to HtrA3 mAbs, also at final concentration of 50 pg/ml) or buffer only, was prepared in 100 mM Tris-HCI (pH 8.0) containing 150 mM NaCl. This mixture was incubated for 50 min at 37 0 C, then 1 pL of freshly prepared p-casein (final concentration 20 pM) (Sigma) was added and incubated for further 10 min at 37 0 C to induce remodelling of the active site (1). Finally, 2.5 pL of peptide substrate (final 30 concentration 0.5 mM) was added and the plates were incubated at 37'C for 6-7 h while the real-time kinetic progression of substrate hydrolysis (release of p-nitroaniline) was monitored every 4 min at 405 nm (Wallac, Victor 2 spectrophotometer, Perkin Elmer, MA). The rate of 4223871_1 (GHMatters) P89027.AU.1 47 substrate hydrolysis (absorbance increase/min) was calculated from the linear phase of the kinetic progression curve and used as the activity unit. In every assay, incubation buffer was used as a blank and other controls included PBS substituted for HtrA3-L and control mAb replaced for HtrA3 mAb. 5 The dose-dependency of the identified inhibitory (10H10) and stimulatory (6G6) mAbs was also tested at a final concentration of 10 and 50 pg/ml respectively. Furthermore, to test whether HtrA3 following 6G6 stimulation could be inhibited by 10H10, HtrA3-L protein was first incubated with 6G6 (50 pg/ml) for 50 min at 37'C, then 10H10 (10 or 50 pg/ml) was added and incubated for further 50 min at 37'C, before the addition of p-casein and the pNA-labelled 10 substrate. To test if HtrA3 following 10H10 inhibition could be activated by 6G6, HtrA3-L was first incubated with 10H10 (50 pg/ml) for 50 min at 37'C, then 6G6 (10 or 50 pg/ml) was added and incubated for further 50 min at 37'C, before the addition of p-casein and the pNA-labelled substrate. Results 15 Identification of mAbs that modulate HtrA3 enzyme activity An in vitro enzymatic activity assay was performed to determine the ability of HtrA3-L to hydrolyse a p-nitroaniline labelled peptide [Ac-VFNTLPMMGKASPV-pNA (SEQ ID NO: 5) as a generic substrate. When HtrA3-L was incubated with the peptide, a progressive release of p-nitroaniline resulting from peptide hydrolysis was detected overtime (Fig 8A), validating the 20 activity assay. To test whether the HtrA3 mAbs could modulate HtrA3 activity, an equal amount of control or HtrA3 mAbs (50 pg/ml) was individually added into the enzyme reaction and the substrate hydrolysis kinetics were monitored. Compared to control mAb, while HtrA3 mAbs 2C4, 9C9 or 3E6 did not significantly affect the HtrA3 activity, 10H10 abolished whereas 6G6 enhanced the protease activity (Fig 8B). This suggested that 10H10 inhibited while 6G6 25 stimulated HtrA3 activity. To further test whether these two activity-modulating mAbs affect HtrA3 activity dose dependently, the reaction was carried out with different concentrations of these mAbs (0, 10 and 50 pg/ml). To illustrate the dose-dependent modulation, the rate of substrate cleavage in the presence of different concentrations of mAbs was expressed as a percentage of the control 30 (containing control mAb at 50 pg/ml). Indeed, 10H10 inhibited (Fig 9), whereas 6G6 stimulated, HtrA3 activity (Fig 10), in a dose-dependent manner. 4223871_1 (GHMatters) P89027.AU.1 48 We next tested if HtrA3 following 6G6 stimulation could be inhibited by 10H 10. HtrA3 protein was first incubated with 6G6 (50 pg/ml) then with 10H10 (10 or 50 pg/mI) before the substrate hydrolysis was monitored. 10H10 inhibited HtrA3 activity after 6G6 stimulation, and the inhibition was dose-dependent (Fig 11). 5 We also tested if HtrA3 following 10H10 inhibition could be activated by 6G10. However, no activity was detected when HtrA3 protein was first incubated with 10H10 (50 pg/ml) then with 6G6 (10 or 50 pg/ml, data not shown). These data confirmed that 10H10 neutralises while 6G6 stimulates HtrA3 enzyme activity. 10 Example 3: Development and validation of HtrA3 ELISAs for Pre-Eclampsia ELISA (the enzyme linked immunosorbent assay) is a powerful assay to detect and quantify a specific protein in a complex mixture. The method enables analysis of protein 15 samples immobilized in microplate wells using specific antibodies. The technique is now a standard method commonly used in medical research laboratories and commercial applications. Among the different types of ELISA, sandwich ELISA is most useful to assay complex protein samples such as serum and plasma because only the specific antigen is immobilized rather than the entire sample of proteins. Sandwich ELISAs require two different antibodies that 20 bind specifically to the antigen (each reacting with a different epitope). The first antibody (bound to the plate) is called the capture antibody or coating antibody, whereas the second antibody (called the detection antibody) detects the immobilized antigen. Such two antibodies are known as "matched pair"; their combination must be identified and validated experimentally in the sandwich ELISA format, because the capture antibody must have the capacity to bind to the 25 targeted proteins in solution and the pair must not compete for binding to the antigen. As such, the identification and validation of the matched capture/detection antibody pairs are the most inventive step in the establishment of a sandwich ELISA. Methods 30 A standard sandwich ELISA protocol (based on Thermo Scientific TECH TIP #65) was used to establish HtrA3-specific ELISAs, using a biotinylated detection antibody and streptavidin-HRP indirect detection system with commonly used reagents and TMB (tetramethyl 4223871_1 (GHMatters) P89027.AU.1 49 benzene) substrate. The major modification was the use of half-area 96-well plates to reduce the sample volume (from 100 pl to 50pl) and other reagents by 50%. Materials and Reagents 5 Clear 96 well plate: Costar® 96 well half area flat bottom high binding polystyrene plate (#3690, Corning, NY, USA). Plate reader: EnVision Multilabel Plate Readers, Perkin Elmer. Coating Buffer: 0.1 M Na 2

CO

3 (carbonate)/ NaHCO 3 (bicarbonate) buffer, pH 9.6. Wash buffer: 0.1 M phosphate, 0.15 M sodium chloride, pH 7.2 containing 0.05% Tween 20. 10 Blocking buffer: 10% (w/v) Bovine Serum Albumin (BSA) in PBS. Capture antibody: HtrA3 mAb 10H10 diluted in Coating Buffer. Detection antibody: biotinylated HtrA3 mAb 9C9 (for the detection of all HtrA3 isoforms) and 6G6 (for the detection of HtrA3-L isoform) diluted in 1/5 strength standard diluent. Standard diluent: 5 mg/ml (w/v) BSA in wash buffer. 15 Serum diluent: 1/4 strength non-pregnant sera (1 in 4 dilution of a non-pregnant serum in standard diluent) to dilute HtrA3 standards and serum samples. Standard: recombinant HtrA3-L-S305A expressed using the wheat-germ system. Enzyme conjugate: Streptavidin-HRP conjugate (#P0397, Dako, Glostrup, Denmark). Substrate: 1-StepTM Ultra TMB-ELISA (#34028, Thermo Scientific, Rockford, IL, USA). 20 Stop solution: 1 M H 2

SO

4 . Procedure Important: Do not allow the plate to dry at any point (1) Dilute the capture antibody in coating buffer to 5pg/ml, add 50 pl to each well, tap plate 25 gently, cover the plate and incubate over night at 4 0 C. (2) Remove the antibody solution and wash plate 3x5 min with 100pl of wash buffer. (3) Add 100 pl of blocking buffer per well, cover the plate and incubate for 1 hr at 37 0 C. (4) Remove the blocking buffer, wash plate 3x with 100pl of wash buffer, add 50 pl of standards and the samples (for serum, 15pl serum diluted in 35 pl standard diluent), cover 30 the plate and incubate for 2 hr at room temperature (RT) on a plate shaker. (5) Remove the solution and wash plate 3x with 1 00pl of wash buffer. (6) Dilute the detection antibodies in diluent: 5mg/ml-2.5mg/ml. (7) Add 50 pl of detection antibody to each well, cover the plate and incubate for 60min at RT. (8) Remove the detection antibody and wash plate 3x with 100pl of wash buffer. 4223871_1 (GHMatters) P89027.AU.1 50 (9) Dilute the Streptavidin-HRP conjugate to the appropriate concentration (1:1000 1:10,000) in diluent. (10) Add 50 pl of the diluted conjugate to each well, cover the plate and incubate for 45 mins at RT. 5 (11) Remove the HRP conjugate and wash plate 4x with 100pl of wash buffer. (12) Add 50 pl substrate solution to each well and develop at RT until desired colour intensity is achieved (normally up to 30min, keep in the dark). (13) Stop the reaction by adding 25 pl of stop solution to each well. (14) Measure the absorbance at 450nm using the plate reader. 10 (15) Masterplex Readerfit software (Hitachi Solutions America, South San Francisco, CA, USA) was used to determine HtrA3 concentrations of unknowns using a standard calibration curve fitted with four parameter logistic (4PL) non-linear regression. Results 15 To select the best matched antibody pairs, all five HtrA3 mAbs were biotinylated and combined with the other four as capture HtrA3 mAbs, and all possible 20 combinations (5x4=20 combinations) were tested on a constant amount of HtrA3 standard. The best combinations (highest signal to background ratio) were: 1OH10 as the capture antibody and 9C9 as the detection antibody for total HtrA3, andlHO0 as the capture antibody 20 and 6G6 as the detection antibody for HtrA3-L isoform. The assays were further optimised by checkerboard titrations (biotinylated mAbs: 5, 1.25, 0.625, 0.312, 0.078ug/ml, Streptavidin HRP: 1:1000, 1:5000, 1:10,000, 1:15,000). The best parameters identified were: 5 pg/ml 1OH10 as the coating antibody, 2.5 pg/ml biotinylated 9C9 and 6G6 as the captuer antibodies, 1:10,000 dilution of Streptavidin-HRP conjugate. 25 The optimised protocols were tested for the detection of wild-type recombinant HtrA3-L and HtrA3-S in the media of COS-7 cells in which HtrA3-L or HtrA3-S was stably transfected (Figure 12). The ELISA for the detection of total HtrA3 (capture: 1OH10, detection: 9C9) detected HtrA3 in the media of cells transfected with both constructs (L6 with HtrA3-L construct, and S11 30 with HtrA3-S) as expected. Likewise, the ELISA for the detection of HtrA3-L isoform (capture: 1OH 10, detection: 6G6) detected HtrA3 in the media of cells transfected with the HtrA3-L specific construct (L6), but not the HtrA3-S construct (S 11). 4223871_1 (GHMatters) P89027.AU.1 51 The assays were also tested for specificity against all mammalian HtrA family members HtrAl, HtrA2, HtrA3-L and HtrA4 (Figure 13). An equal amount of purified recombinant HtrA proteins (90 pM) were subjected to HtrA3 ELISAs, both ELISAs detected HtrA3 only, but did not detect other family members (HtrAl, HtrA2 or HtrA4). 5 We observed different-shaped curves (Figure 14) for the HtrA3 standards when diluted in the standard diluent vs serum diluent, implying that HtrA3 standards need to be diluted in the serum diluent when assaying serum samples for both HtrA3 ELISAs. The assays were then applied to detect HtrA3 isoforms in pregnant human sera, by applying 50 uL of diluted sera (1 in 4 dilution) collected from pregnant women across gestation 10 (9, 20 and 34 weeks of gestation). The detected HtrA3 pattern (Figure 15) across gestation is highly consistent with our previous publications using Western blot (Li et al 2011 J Clin Endocrinol Metab 96:403-411). The best antibody pair combinations for HtrA3 ELISAs are summarized in Table 4. 15 4223871_1 (GHMatters) P89027.AU.1

Claims (22)

1. A monoclonal antibody (mAb) and antigen binding fragment thereof that is, or binds competitively with, an antibody produced from hybridoma cell line 1OH10 deposited with 5 CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019, hybridoma cell line 3E6 deposited with CellBank Australia as accession number CBA20120017, hybridoma cell line 2C4 deposited with CellBank Australia as accession number CBA20120015 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018. 10

2. The antibody of claim 1 which binds HtrA3 but not HtrA1 or HtrA2.

3. The antibody of claim 1 which binds a HtrA3 short isoform and a HtrA3 long isoform but not HtrA1 or HtrA2, in which the antibody is produced by hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019 and hybridoma cell line 2C4 15 deposited with CellBank Australia as accession number CBA20120015.

4. The antibody of claim 1 that binds a HtrA3 long isoform but not a HtrA3 short isoform or HtrA1 or HtrA2, in which the antibody is produced hybridoma cell line 3E6 deposited with CellBank Australia as accession number CBA20120017 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018. 20

5. An antagonist monoclonal antibody for HtrA3, which antibody is produced from hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016.

6. The antibody of claim 5 which is neutralising for HtrA3.

7. An agonist antibody monoclonal antibody for HtrA3, which antibody is produced from 25 hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018.

8. The antibody of any preceding claim, conjugated to acceptor beads, or coated onto ELISA plates.

9. The antibody of any one of claims 1 to 8 in which the antibody is biotinylated.

10. A kit comprising a monoclonal antibody or antigen binding fragment thereof that 30 specifically binds to the same epitope as a monoclonal antibody produced by hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019, hybridoma cell line 3E6 deposited with CellBank Australia as accession number CBA20120017, hybridoma 4223871_1 (GHMatters) P89027.AU.1 53 cell line 2C4 deposited with CellBank Australia as accession number CBA20120015 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018.

11. A kit for HtrA3 short isoform and a HtrA3 long isoform but not HtrA1 or HtrA2, the kit comprising an antibody produced by hybridoma cell line 1OH10 deposited with CellBank 5 Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019 and hybridoma cell line 2C4 deposited with CellBank Australia as accession number.

12. A kit for HtrA3 long isoform but not a HtrA3 short isoform or HtrA1 or HtrA2, the kit comprising an antibody produced hybridoma cell line 3E6 deposited with CellBank Australia as 10 accession number CBA20120017 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018.

13. Use of a monoclonal antibody or antigen binding fragment thereof that specifically binds to the same epitope as a monoclonal antibody produced by hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 15 deposited with CellBank Australia as accession number CBA20120019, hybridoma cell line 3E6 deposited with CellBank Australia as accession number CBA20120017, hybridoma cell line 2C4 deposited with CellBank Australia as accession number CBA20120015 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018 for detecting the presence or amount of HtrA3 in a biological sample. 20

14. Use of a monoclonal antibody or antigen binding fragment thereof that specifically binds to the same epitope as a monoclonal antibody produced by hybridoma cells line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019, hybridoma cell line 3E6 deposited with CellBank Australia as accession number CBA20120017, hybridoma cell line 2C4 25 deposited with CellBank Australia as accession number CBA20120015 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018 in the diagnosis of diseases involving dysregulation of HtrA3 such as preeclampsia and cancer, particularly lung cancer.

15. An assay for detecting HtrA3 in a serum or plasma or blood or urine sample, the method 30 comprising contacting the sample with at least one antibody or fragment thereof produced by hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019, hybridoma cell line 3E6 deposited with CellBank Australia as accession number 4223871_1 (GHMatters) P89027.AU.1 54 CBA20120017, hybridoma cell line 2C4 deposited with CellBank Australia as accession number CBA20120015 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018 and detecting binding of the antibody or fragment thereof to HtrA3.

16. A method for detecting a disease involving dysregulation of HtrA3, the method 5 comprising assaying for HtrA3 using the assay of claim 15.

17. A method of producing a hybridoma cell line that expresses a binding protein for HtrA3 but not HtrA1 or HtrA2 comprising the steps of: a) immunising a BALB/C mouse with an antigen comprising HtrA3- L-S305A or amino acids 230-239 of HrtA3-L and HtrA3-S (TIKIHPKKKL SEQ ID NO: 4) for a time and under conditions sufficient for the mouse to produce antibodies 10 against the antigen; b) harvesting and purifying cells from the spleen of the mouse; c) fusing the spleen cells with myeloma cells in order to produce hybridomas; and d) selecting a hybridoma cell line which expresses the binding protein which bind either or both of HtrA3-L and HtrA3-S but not HtrA1 or HtrA2.

18. Cells of the hybridoma cell line 1OH10 deposited with CellBank Australia as accession 15 number CBA20120016, hybridoma cell line 9C9 deposited with CellBank Australia as accession number CBA20120019, hybridoma cell line 3E6 deposited with CellBank Australia as accession number CBA20120017, hybridoma cell line 2C4 deposited with CellBank Australia as accession number CBA20120015 and hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018. 20

19. A method of treating cancer comprising administering an agonist antibody to HtrA3 which antibody is produced from hybridoma cell line 6G6 deposited with CellBank Australia as accession number CBA20120018.

20. The method of claim 19 in which the cancer is but not limited to lung cancer, ovarian cancer or endometrial cancer. 25

21. A method of treating preeclampsia or other diseases involving dysregulation of HtrA3 comprising administering an antagonist antibody to HtrA3 which antibody is produced from hybridoma cell line 1OH10 deposited with CellBank Australia as accession number CBA20120016.

22. The method of claim 21 in which the antibody is neutralising for HtrA3. 30 4223871_1 (GHMatters) P89027.AU.1