AU726994B2 - PAPP-A, its immunodetection and uses - Google Patents
PAPP-A, its immunodetection and uses Download PDFInfo
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- AU726994B2 AU726994B2 AU78548/98A AU7854898A AU726994B2 AU 726994 B2 AU726994 B2 AU 726994B2 AU 78548/98 A AU78548/98 A AU 78548/98A AU 7854898 A AU7854898 A AU 7854898A AU 726994 B2 AU726994 B2 AU 726994B2
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- papp
- antibody
- monoclonal antibody
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- pregnancy
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Description
Vt
I
AUSTRALITA
Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT
S.
S 5
S
*5
S.
S
5* S Applicants: NORTHERN SYDNEY AREA HEALTH SERVICE and MICHAEL JOSEPH SINOSICH Invention Title: PAPP-A, ITS IMMIJNODETECTION AND USES *5 I S S.
S.
The following statement is a full description of this invention, including the best method of performing it known to us: 1A PAPP-A, ITS IMMUNODETECTION AND USES Technical Field The present invention relates to: purified pregnancy-associated plasma protein-A (PAPP-A); PAPP-A variants; polynucleotides encoding PAPP-A; isolation and purification of PAPP-A; monoclonal antibodies raised against PAPP-A; use of these monoclonal anti-bodies and PAPP-A for diagnostic purposes, including a kit for the assaying of PAPP-A levels in a sample; use of PAPP-A measurement, in conjunction with another marker of trophoblastic activity (such as chorionic gonadotrophin or its subunits), to discriminate between feto-placental abnormalities, such as Trisomy 21 (Down Syndrome), and oncological status, such as Gestational Trophoblastic 15 Diseases (GTD); use of PAPP-A monoclonal antibodies to isolate fetal trophoblast cells for prenatal fetal cytogenetic diagnosis; use of PAPP-A, as a target antigen for active immunological contraception, and PAPP-A antibodies as a passive contraceptive vaccine; use of 20 PAPP-A as a medicament and a medicament comprising an effective amount of PAPP-A.
BACKGROUND
ART
Placental proteins are those proteins expressed during pregnancy by the human placenta. The ability to detect the presence and concentrations of these proteins has the potential to provide a reliable diagnostic marker of fertilisation, implantation and pregnancy prognosis.
A number of placental proteins have now been isolated and at least partially characterised. These include human chorionic gonadotropin (hCG), pregnancyspecific 91 glycoprotein (SP1), placental protein early pregnancy factor (EPF), and pregnancyassociated plasma protein-A (PAPP-A) These proteins are detectable, in maternal blood, at various stages during pregnancy. For example, EPF activity is detectable within 24 hours after conception.
HCG is measurable just after implantation, at about 9 to 11 days post-ovulation, SP1 is detectable from 18 to 23 2 days post-ovulation. In singleton pregnancies, PAPP-A can be detected approximately 28-32 days post-ovulation 2 Placental proteins are also detectable for varying periods during pregnancy. For example, EPF is detectable at least for the first half of pregnancy, whereafter activity declines until it is totally absent during the third trimester in some women. HCG levels rise rapidly to peak at about 8 to 12 weeks gestation. The levels of SP1 rise exponentially with peak concentrations being reached at term pregnancy. Like SP1, PAPP-A concentrations also rise exponentially in the first trimester of pregnancy to peak at term 2 Whilst it has been suggested to measure the presence of placental proteins for early detection of pregnancy 15 (for example, see European Application 316919), there is a growing body of documented evidence that at least some placental proteins, particularly PAPP-A, may be used to predict pregnancy viability, including early pregnancy failure, extra-uterine gestations, aneuploid and/or 20 abnormal pregnancies, such as Down's Syndrome 3 and Cornelia de Lange Syndrome 4.
PAPP-A, first described almost two decades ago 1 is a large zinc containing glycoprotein, rich in carbohydrate, with many physicochemical similarities to a 2-macroglobulin 5 It has been detected in maternal circulation 5 pre-ovulatory ovarian follicular fluid 6 in seminal plasma 7 and blood of patients with trophoblastic disease 8 PAPP-A is a homotetramer, with each monomeric subunit having a molecular weight of approximately 200 kDa. The subunits are linked by disulphide bonds to form dimers of approximately 400 kDa. Native PAPP-A consists of two dimers linked by Van der Waals (ionic) forces.
Native PAPP-A has a molecular weight of approximately 820 kDa, regardless of whether it is derived from follicular fluid, seminal plasma, oncological or normal placental tissue 7 The mature protein has a 2-61 electrophoretic mobility, with an isoelectric point of approximately
I"
3 4.2-4.5 It is a non-competitive and potent inhibitor of human granulocyte elastasel 0 It has been suggested that the biological function of PAPP-A is to act as a local protective barrier against host (maternal) phagocytic-proteolytic defences to either inseminated sperm or the developing feto-placental unit 1 This may be due to PAPP-A forming a proteetive sheet around the chorionic villus at the utero-placental interface 12 Disruption of this protective layer may explain the correlation between depressed PAPP-A levels and pregnancy failure. PAPP-A may also play a role in zinc homeostasis 5 Schindler and Bischof 13 suggested that the protein was ubiquitous and, therefore, of little practical use in 15 pregnancy viability diagnosis. 14 15 However, it was subsequently shown that these results were due to impure PAPP-A isolates, due to the difficulties in isolating PAPP-A free of a 2-macroglobulin, and polyspecific 2 antisera 16 20 Sinosich et al.
3 first suggested that a depressed or undetectable PAPP-A level in maternal blood was diagnostic of pregnancy failure. Later, Sinosich et al.
17 showed that, of five successful in vitro fertilisation volunteers, three patients with normal pregnancy outcome 25 had circulating PAPP-A levels within the 80% confidence limits of the normal range. By contrast, circulating PAPP-A levels in the patient who spontaneously aborted at seventeen weeks were below the tenth percentile throughout the entire gestation. In the fifth patient, who had a ruptured ectopic pregnancy, PAPP-A could not be detected at any stage- during the pregnancy. These findings were complimented by Westergaard et al. (1983) 18 who reported that, in a sample group of 51 patients, who conceived spontaneously, with vaginal bleeding in the first half of pregnancy, concentrations of PAPP-A were consistently lower in pregnancies which failed.
Similarly, Sinosich et al. (1985) 19 showed that, in a group of 21 women who conceived by in vitro 4 fertilisation, PAPP-A levels were consistently depressed, for many weeks, in those women whose pregnancies failed.
The same group showed that, of forty seven serum samples obtained from patients with a tubal pregnancy, only two were positive for PAPP-A, indicating that severely depressed or undetectable serum PAPP-A levels were an aid in the diagnosis of extra-uterine pregnancy.20 In 1990, Brambati et al.
21 reported that first trimester maternal serum concentrations of PAPP-A were low in pregnancies associated with Down's syndrome.
Later, Wald et al. (1992) 3 confirmed that PAPP-A concentration was significantly lower in women with Down's syndrome pregnancies compared to PAPP-A levels in a control group of normal pregnancies.
15 It has also been reported that PAPP-A was detected in the circulation of patients with hydatiform mole 2 suggesting a potential role for PAPP-A quantification in diagnosis and management of certain tumours.
These findings demonstrate the potential diagnostic 20 value of measuring PAPP-A levels for monitoring fetoplacental status. Moreover, in view of the increasing use of in vitro fertilisation techniques, and the relatively high proportion of early pregnancy failures associated with these techniques, the measurement of 25 PAPP-A levels to monitor pregnancy viability and thereby minimise patient trauma is clinically advantageous.
Lin. et al. (1974) 22 described the use of an electroimmunoassay to measure PAPP-A levels in advanced pregnancy. This assay was insensitive and limited to latter stages of pregnancy. Sinosich et al. (1982) 2 and (1984) 6 described the first sensitive radioimmunoassay (RIA) which detected PAPP-A in serum obtained from first trimester pregnancies. This assay used radioactively labelled purified PAPP-A together with rabbit anti-human PAPP-A antiserum. The sensitivity of this RIA (2.9 Ag/L) enabled PAPP-A detection in maternal blood after the first six weeks of pregnancy. The assay made it possible to detect PAPP-A in other fluids (amniotic fluid, seminal 5 plasma, follicular fluid, gestational trophoblastic disease, culture media), previously beyond the limits of detection. This assay also made it possible to study the kinetics and physiology of maternal PAPP-A levels in the first trimester of pregnancy, a crucial stage for fetoplacental development.
The PAPP-A RIA differed from standard protocols in that molecular size of tracer 1 2 5 I-PAPP-A; Mr 820kDa) and immune complexes required modification of the separation phase. Optimal separation of antibody-bound from antibody-free tracer was achieved with second antibody polyethylene glycol (PEG) solution, in the ratio of 2:1, 2nd antibody-PEG to assay reaction volume. Under these conditions, assay blank values could be reduced to 15 whilst maximum binding would approach 60-70%.
The development of sensitive and reliable techniques for measuring PAPP-A is dependent on being able to isolate the protein in a sufficiently pure form and/or the generation of monospecific antibodies.
A number of methods for isolating and purifying PAPP-A have been described previously. For example, Lin.
et al. (1974)23 describe a procedure based on classical protein fractionation technology. This procedure utilised: S* 25 i) solubility; ii) charge; and iii) size.
This procedure was technically cumbersome and resulted in a low yield of impure material.
Bischof (1979) 2 4 described a method of isolating and purifying PAPP-A. This procedure utilised: i) solubility; ii) charge; iii) lectin affinity; iv) size fractionation; and v) negative immunoaffinity.
This was even more cumbersome than the earlier procedure. The final product was still impure and yields 6 remained low.
Sutcliffe et al.(1979) 2 5 and Folkersen et al.
(1981) 2 6 described a purification method which used; i) solubility; ii) positive immunoaffinity chromatography (immobilised anti-PAPP-A antibodies); and iii) size or charge, respectively.
Step (ii) is detrimental to PAPP-A integrity and neither procedure resulted in pure PAPP-A. Although the final yield was improved by this method, the quality of the protein yield was sacrificed.
Based on an interaction with heparin (Sinosich et al. 1981) 2 7 Sinosich et al. (1982) 1 0 reported the first application of heparin-Sepharose for PAPP-A purification.
15 For the first time it was possible to prepare a high yield of highly purified PAPP-A. However, by more current and stringent criteria, this preparation was also found to be impure.
Disclosure of the Invention 20 The inventor has developed a purification procedure which enables PAPP-A to be purified to a much higher degree than achieved with prior art purification techniques and in particular to a level which permits the amino-acid sequencing of the protein. The inventor has 25 sequenced the first thirteen (13) N-terminal amino acids of human PAPP-A and this sequence is: Glu-Ala-Arg-Gly-Ala-Pro-Glu-Glu-Pro-Ser-Pro-Pro-Ser According to a first aspect of the present invention there is provided the protein PAPP-A, substantially free of other (primate) proteins, said PAPP-A being a homotetramer having a molecular weight of approximately 820 kiloDaltons and an isoelectric point of approximately each monomeric subunit of the homotetramer having a molecular weight of approximately 200 kiloDaltons and an N-terminal amino-acid sequence of Glu-Ala-Arg-Gly-Ala- Pro-Glu-Glu-Pro-Ser-Pro-Pro-Ser.
The ability to sequence the protein indicates that the preparation is at least 90% pure.
7 Biologically active proteins are usually only present in trace amounts in biological systems.
Consequently, purification from naturally occurring sources is expensive and time consuming. Having purified PAPP-A to sufficient purity to obtain amino acid sequence for the protein, it is now possible for the first time to produce degenerate nucleotide probes which colrespond to the determined amino acid sequence. These probes can then be used to detect nucleic acid encoding PAPP-A and clone it. At this level of purity PAPP-A can also be used as an antigen to raise antibodies to detect
PAPP-A
expressing clones.
Production of the PAPP-A protein by recombinant techniques and isolation of the DNA encoding PAPP-A also 15 makes it possible to obtain a complete amino acid sequence for PAPP-A. The complete amino acid sequence can be used to determine appropriate sites for mutagenesis in the production of clinically useful PAPP-A *variants.
20 According to a second aspect of the present invention there is provided a PAPP-A variant. Variants of PAPP-A in accordance with this invention are polypeptides which correspond to or comprise a portion of PAPP-A or have homology with the PAPP-A amino acid 25 sequence.
For the purposes of this description "homology" between two peptide sequences connotes a likeness short of identity, indicative of a derivation of the first sequence from the second. In particular, a polypeptide is "homologous" to PAPP-A of the invention if a comparison of amino-acid sequences between the polypeptide and PAPP-A reveals an identity of greater than about 70%. Such a sequence comparison can be performed via known algorithms, which are readily implemented by computer.
According to a third aspect of the invention there is provided a polynucleotide encoding PAPP-A or a PAPP-A variant of the present invention.
4 8 a a a a.
a a. a a a. a.
a a. a According to a fourth aspect of the invention, there is provided a method for isolating and purifying PAPP-A, said method comprising: applying the sample to a positive affinity chromatography on heparin-Sepharose; (ii) size fractionating the fractions obtained from step on a gel filtration column; and (iii) applying the fractions obtained from step (ii) to an anion exchange column; and (iv) applying the fractions obtained from step (iii) to a matrix with immobilized antibodies.
According to a fifth aspect of the invention, there are provided monoclonal antibodies which recognise 15 PAPP-A. Typically the monoclonal antibodies of this aspect recognise PAPP-A but do not significantly crossreact with immobilized heparin-Sepharose binding proteins extracted from normal male serum. Preferably the monoclonal antibodies are specfic for PAPP-A. In 20 particular, the inventor has developed five monoclonal antibodies which specifically recognise five epitopes of the PAPP-A protein.
The first monoclonal antibody clone 25-1 has been isotyped as IgG 2 (K light chain) with an affinity 25 constant of 1.26 x 10 9 Clone 25-1 reacts specifically with human PAPP-A but not with chimpanzee or rhesus
PAPP-A.
The second monoclonal antibody Clone 27-66, isotyped as IgM with K light chain specifically recognises PAPP-A- from human, chimpanzee and rhesus primates.
The third monoclonal antibody Clone 5-62, isotyped as IgG 1 with K light chains with an affinity constant of 2.65 x 10 9 reacts specifically with human and chimpanzee PAPP-A but not with rhesus PAPP-A.
The fourth monoclonal antibody Clone 18-9, isotyped as IgG with K light chains, reacts specifically with human and chimpanzee PAPP-A but not with rhesus 9
PAPP-A.
The fifth monoclonal antibody Clone 41.1, has yet to be isotyped and characterised.
The advantages conferred by the development of monoclonal antibodies to PAPP-A are that they are specific for a distinct epitope. This specificity allows the development of an assay with increased, sensitivity and reliability. This increased sensitivity permits the detection of PAPP-A in the circulation within the first six weeks of normal pregnancy and depressed levels in abnormal pregnancies.
The use of monoclonal antibodies also permits the development of immunoassays which are independent of labelled antigen (PAPP-A). The term "immunoassays", as 15 used herein, includes any method for PAPP-A detection mediated by the use of antibodies (classes IgG, IgA, IgD, IgE or IgM) or derivatives thereof (including Fab and F(ab) 2 fragments). Antibodies may be polyclonal or monoclonal, and may be generated by active immunisation 20 of a host animal (including mouse, rat, guinea pig, rabbit, sheep, horse, donkey or other mammalian species), hybridoma technology and/or molecular biology for production of recombinant antibodies. Quantitative and qualitative PAPP-A detection may be achieved in liquid and/or solid phase and, in addition to immunoassays, includes in situ hybridisation and immunohistochemistry, by way of example.
Monoclonal antibodies also allow the development of sandwich assays with greater amplification potential.
The single epitope specificity of the antibodies permits the detection of PAPP-A active sites and, therefore, quantification of bio-active
PAPP-A.
According to a sixth aspect of the present invention, there is provided a method for the detection of PAPP-A in a sample using a monoclonal antibody of the fifth aspect to detect PAPP-A. Typically, the method comprises the steps of:contacting the sample with a PAPP-A capture S:22604E 10/10/95 10 phase which consists of one or more of the following: monoclonal PAPP-A antibody; polyclonal PAPP-A antibody; immobilised heparin; immobilised divalent metal cations (Cu Zn+, Co immobilized lectins (Concanavalin- A, Helix pomatia, Lens culinaris, Limulus polyphemug, phytohaemaglutinin, Ricinus co4munis, Wheat 0 germ, or others with equivalent specificities), and, other specific (such as receptors) or nonspecific ligands (such as dyes) with which
PAPP-
A has an affinity.
(ii) contacting the immobilised or captured PAPP-A sample with a monoclonal antibody, of the fifth aspect of the invention, labelled with a detectable marker; (iii) incubating the sample and the labelled 20 antibody to permit the labelled antibody to bind to any PAPP-A in the sample and; (iv) detecting the labelled antibody.
Typical methods include enzyme immunoassays (EIAs) or immunoradiometric assay (IRMA) formats.
25 According to a seventh aspect of the present invention there is provided a kit for the detection of PAPP-A in a sample which kit comprises: at least one monoclonal antibody according to the fifth aspect of the present invention together with a positive and/or negative control. The PAPP-A assay kit, developed for manual and/or automated application, is not limited to human application. Selection of appropriate polyclonal-monoclonal PAPP-A antibody combination can extend application to sub-human primates.
The ability to produce large quantities of recombinant PAPP-A or variants thereof permits use as a medicament for the treatment of pregnant women with low or zero PAPP-A levels in order to increase the likelihood of the pregnancy proceeding successfully to term.
S:22604E 10/10/95 -I 11 According to an eighth aspect of the present invention, there is provided the use of PAPP-A as a medicament together with a pharmaceutically acceptable excipient to treat a pregnant patient exhibiting the absence of or low levels of PAPP-A.
Formulation of PAPP-A for this purpose with standard carriers, excipients and diluents is performed in accordance with standard pharmaceutical techniques.
According to a ninth aspect of the present invention there is provided a medicament comprising an effective amount of PAPP-A together with a pharmaceutically acceptable carrier, diluent or excipient.
According to tenth aspect of the present invention, there is provided the use of PAPP-A as a target for 15 immunological contraception. Immunological fertility regulation may be achieved by active vaccination against intact PAPP-A or fragments thereof. Active vaccination may be achieved by any of the accepted oral, mucosal or subcutaneous routes (of antigen administration) used to induce an immune response in humans. The PAPP-A antigen may be chemically modified, genetically engineered or the genome inserted into a vector to enhance immungenecity when expressed in the host.
Administration of PAPP-A antibodies (or fragments thereof) may be applied for fertility regulation by passive immunisation or for immunolocalisation and/or immunoneutralisation of trophoblastic tumors or management of extrauterine pregnancies. Antibody administration can be achieved by any of the procedures outlined for PAPP-A antigen administration.
According to the eleventh aspect of the present invention, there is provided the use of PAPP-A (polyclonal and monoclonal) antibodies for isolation of trophoblast cells from systemic maternal circulation or reproductive tract (vaginal cavity, cervical canal, cervical os). The isolated trophoblast cells may be cultured and applied for prenatal fetal karyotyping.
According to the twelth aspect of the present 12 invention, there is provided the use of PAPP-A immunodetection to discriminate between normal and abnormal pregnancies. Abnormal pregnancies include multiple gestations (more than one fetus), extra-uterine implantation, anembryonic pregnancies, death in utero, incomplete miscarriage, spontaneous abortion, fetal malformations and aneuploidies, as examples. Quantitative or qualitative PAPP-A immunodetection may be performed on maternal biological fluids, such as blood. Blood may be collected by routine venepuncture, finger pricking or by any accepted medical procedure. The blood may be anticoagulated or coagulated, to permit the removal of supernatant for PAPP-A analysis. The blood may be stored frozen or dried onto an inert absorptive medium, such as 15 filter paper, for transportation, subsequent extraction and analysis.
According to the thirteenth aspect of the present invention, there is provided the use of PAPP-A immunodetection in female biological fluids, such as 20 ovarian follicular fluid and reproductive tract secretions, to assess folliculogenesis, granulosa cell status and ovulation.
According to the fourteenth aspect of the present invention, there is provided the use of PAPP-A 25 immunodetection for diagnosis and management of gestational trophoblastic disease (GTD) In hydatidiform mole, a benign GTD exhibits a molar expression ratio of PAPP-A to hCG (or free b-subunit) which is indistinguishable from that observed in normal pregnancy.
As the GTD transforms into the more aggressive and invasive choriocarcinoma, the molar expression ratio of PAPP-A to hCG (or free b-subunit) decreases, that is hCG (or free b-subunit) expression is maintained or increased, whereas, PAPP-A expression is downregulated.
This trophoblast antigen expression ratio can be applied as an algorithm for diagnosis and management of GTD.
According to a fifteenth aspect of the invention, PAPP-A antibodies are used for the immuno-treatment of S:22604E 10/10/95 13 trophoblastic tumors.
According to a sixteenth aspect of the present invention, there is provided the use of PAPP-A immunodetection in male reproductive tract secretions, such as seminal plasma and prostatic fluid, to assess the clinical state of male accessory glands, such as prostate gland, of the reproductive tract.
According to a seventeenth aspect of the present invention, there is provided the use of PAPP-A immunodetection for specific discrimination of pregnancies carrying Trisomy 21 (Down Syndrome) fetuses from normal and abnormal pregnancies. Although both hCG (and free b-subunit) and PAPP-A are trophoblast proteins, in this particular clinical situation their respective expression rates are discordant. Whereas PAPP-A expression is downregulated, hCG (and free b-subunit) expression is upregulated. Therefore, the molar expression of PAPP-A to hCG (or free b-subunit) can be applied as an algorithm for prenatal screening of Down 20 Syndrome pregnancies in early gestation.
Brief Description of the Drawings The invention will now be described with reference to the following drawings: Figure 1 The N-terminal amino acid sequence of 25
PAPP-A.
Figure 2 Anion exchange on Mono Q. Purification of primate (human, A; macaque, B) PAPP-A by high pressure liquid chromatography anion exchange on Mono Q. Dashed lines represent increases in NaCl concentration to effect PAPP-A desorption (at 46-48 min).
Figure 3 Assay protocol for PAPP-A quantification consists of a capture phase immobilized on an inert support and a monoclonal antibody for quantification. The monoclonal antibody may be tagged or visualized with a secondary antibody. The latter option S:22604E 10/10/95 -If 14 Figure 4 Figure 5 Figure 6 Figure 7 0466 00 a 1 0 *000 00 0 Figure 8 enhances signal amplification and assay sensitivity.
Influence of capture antibody concentration on PAPP-A standard curves.
Influence of temperature on the binding equilibrium of immobilised immunoglobulin at 4 0 C, room temperature and 37° 0 C, Maximal PAPP-A capture was achieved after overnight incubation (15 hours).
Binding equilibrium of monoclonal antibody clone 25-1 at various concentrations.
Binding equilibrium was achieved by 4 hours at room temperature.
Standard curves for PAPP-A dose response at two dilutions for clone 25-1.
Logit-log graphs for human serum and normal and caesarian placental homogenates assayed by RIA with clone 25-1. The results suggest parallelism between circulating and tissue PAPP-A.
Logit-log by graphs for human serum and normal and caesarean placental homogenates assayed by EIA with clone 25-1. The results again suggest parallelism between circulating and tissue PAPP-A.
Logit-log graphs for human, chimpanzee and rhesus placental homogenates assayed by EIA with clone 27-66. The results indicate that clone 27-66 equally reacts with PAPP-A from all three species.
Logit-log graphs for human, chimpanzee and rhesus placental homogenates assayed by EIA with clone 5-62. The results indicate that clone 5-62 reacts with PAPP-A from human and chimpanzee but not rhesus.
Logit-log graphs for human, chimpanzee and rhesus placental homogenates assayed by EIA with clone 25-1. The results indicate Figure 9 Figure 10 Figure 11 Figure 12 15 that clone 25-1 reacts only with human
PAPP-A.
Table 1 Monoclonal 5-62 and 25-1 were tested for competition against a variety of antisera.
Table 2 The results indicate that the monoclonals recognise different epitopes.
Best Mode for Carrying out the Invention Methods of recombinant PAPP-A production and preparation of variants are performed in accordance with standard techniques as taught, for instance, in Maniatis et al. (1984) 31 which is herein incorporated by reference.
Formulation of PAPP-A medicaments is peformed in accordance with standard pharmaceutical techniques.
Where recombinant techniques are used for the 15 preparation of the protein of the invention, the protein can be prepared by constructing degenerate DNA probes coding for amino acid sequence of PAPP-A as described above and using these probes to isolate positive clones from c-DNA libraries according to standard techniques as 20 found in text books such as Maniatis et al. (1984) 31 These positive clones are then incorporated into appropriate vectors with expression cassettes according o* to standard techniques. These vectors are then used to transform host cells to allow the production of the 25 recombinant PAPP-A protein.
The selection of appropriate vectors, hosts and expression strategies can be performed in accordance with standard techniques of molecular biology.
Suitable expression systems include, for instance, Chinese hamster ovary expression systems.
It will be readily apparent to a skilled artisan that where PAPP-A is produced recombinantly it may not be necessary to purify the protein before use. Recombinant production can be conducted in well-defined prokaryotic and eukaryotic hosts. Production in non-human systems yields the protein in a form at least substantially free of other human proteins.
The homologous polypeptides can be produced by S:22604E 10/10/95 16 conventional site-directed mutagenesis, which is one avenue for routinely identifying residues of the molecule that can be modified without rendering the resulting polypeptide biologically inactive, or by chemical synthesis.
Those variants which correspond to or comprise a portion of PAPP-A of the invention without being coincident with PAPP-A of the invention, within the scope of the invention, are those molecules which retain the immunogenic or biological activity of the native PAPP-A protein.
These variants may be prepared synthetically by peptide synthesis techniques, recombinantly or by cleavage from an isolated protein of the invention.
Methods of isolating, characterising and cloning polynucleotides encoding PAPP-A using probes constructed from the N-terminal amino acid sequence are performed in accordance with standard techniques as taught, for instance, in Maniatis et al (1982) 31 Isolation and Purification of PAPP-A The protocol consists of five chromatographic procedures and is applicable to human and non-human PAPP- A species. The protocol can be used to isolate PAPP-A .from any biological fluid, culture medium or tissue 25 extract.
1. Positive Affinity Chromatography on Heparin-Sepharose: The column was equilibrated with aqueous buffers, such as 50mM Tris-HCl, pH 7.4-7.8, containing 150 mM NaCl (TBS 0.15M NaCI). Sample application (iml/min) resulted in only 11.3% of applied serum proteins being bound to the matrix. The remaining proteins do not interact with heparin and are easily removed from PAPP-A.
By increasing the ionic strength of equilibration buffer to BS 0.3M NaCl, the low affinity heparin interaction was reduced and only 0.5% of applied proteins were retained by the ligand (heparin). Although PAPP-A recovery was 100%, the yield was 39% for a S:22604E 10/10/95 17 purification factor of 486.
Heparin-PAPP-A interaction is heterogeneous, with minor amounts of PAPP-A not interacting with heparin (Sinosich 1985) 27 Although the significance of this heterogeneity is uncertain, it is clearly not related to heparin. Therefore, the heterogeneity resides with PAPP-A and may be related to PAPP-A isoforms or metabolic clearance, and, hence, provide a distinction between functional and inactive PAPP-A.
Desorption of matrix bound PAPP-A was achieved with application of high ionic strength buffers, such as TBS containing 0.6M NaCl or greater (up to 2.0M NaCl).
PAPP-A elutes as a sharp peak and is concentrated into 3 or 4 fractions, dependent on column size and fraction 15 volume. Desorption may also be achieved by applying the heparin antagonist, protamine sulphate, but matrix regeneration is more complex.
2. Size Fractionation: PAPP-A containing fractions were pooled (36ml) and applied onto a 5 x 90cm column (Vol=1.711) packed with gel filtration matrix Ultragel AcA34 or AcA22 or equivalent. PAPP-A containing fractions were pooled and dialysed against 20 mM TBS, containing 10 mM NaCI, pH 7.4-7.8. PAPP-A recovery for this procedure was 98.8% 25 with an overall purification factor of 827.
3. Anion Exchane (Mono 0): The dialysed PAPP-A pool (110 ml) from size fractionation was applied onto a 10 ml anion exchange column, such as Mono Q, by high pressure liquid chromatography. The PAPP-A containing pool from size fractionation was applied in 10 ml aliquots at Iml/min.
After 20 min., buffer ionic strength was increased to NaCI by a gradual linearly increasing concentration gradient in NaCl. This removed low affinity interactions. The high affinity PAPP-A interaction was dissociated with a stepwise increase in NaCl concentration to TBS-IM NaCI (Figure 2).
18 4. Negative Immunoaffinity Chromatography: Rabbit antibodies against serum proteins, heparin binding serum and tissue proteins were immobilised onto inert particles such as CNBr-Sepharose.
PAPP-A
containing fractions (25 ml) from step 3 (above) were repeatedly passed over this matrix to remove any contaminants.
Positive Affinitive Chromatography: This is optional, but provides an easy means of concentrating PAPP-A from larger into smaller volumes prior to aliquotting and storage. PAPP-A pool (90 ml), from step 4 was applied onto a .120 ml Heparin-Sepharose column. Matrix bound PAPP-A is desorbed with TBS-1M NaCl for a final PAPP-A yield of up to 22%, for an overall 15 purification factor of 1483.
6. The urity of PAPP-A preparation was assessed and confirmed by: i) SDS-PAGE analysis (Sinosich et al., 1990) 28 ii) radioimmunoassay (Sinosich and Zakher, 199129; Sinosich et al. 19822, 19846) and iii) NH 2 -terminal amino acid sequencing.
The NH 2 -terminal amino acid sequence for PAPP-A was sequenced by the School of Biological Sciences, Macquarie Unversity according to standard techniques and was 25 determined to be: Glu-Ala-Arg-Gly-Ala-Pro-Glu-Glu-Pro-Ser-Pro-Pro-Ser- As there is only one NH 2 -terminal amino acid sequence detected, this indicates both that PAPP-A preparation is free of contamination with other proteins, and PAPP-A subunits are identical.
As this sequence is unique to PAPP-A, immunodetection of PAPP-A means the detection of a protein or proteins containing this NH 2 terminal amino acid sequence.
Monoclonal Production Immunisation: Pure PAPP-A, isolated from pooled late pregnancy serum was used to immunise BALB/c mice by two protocols.
19 PAPP-A (30Ag) in 0.05M sodium phosphate buffer, pH 7.4, containing 150 mM NaCi (50 pl; PBS) was emulsified in 50 Il Complete Freund's Adjuvant (CFA) and injected into the footpads of mice. Fourteen (14) days later, a booster dose of 65 Ag PAPP-A/100 il PBS was injected into the thigh muscle of Mouse 1. This mouse was sacrificed days later for hybridoma cell fusion.
Mouse 2 was subcutaneously immunised with 130 Ag PAPP-A in CFA, followed, three weeks later, with an intraperitoneal injection of 150 Ag PAPP-A in Incomplete Freunds Adjuvant (ICA). One week later the mouse was boosted with an intravenous injection of 65 Ag PAPP-A and the animal sacrificed five days later.
Hybridomas were prepared by fusing splenocytes and inguinal lymph node cells with X63 Ag 8.6.5.3. myeloma cells (Gaefee et al. 1977) and cultured in HAT medium.
Once established, the selected hybridomas were cultured in aminopterin-free HT medium. Antibody production was monitored by enzyme immunoassay (EIA) and antibody producing cells were cloned by limiting dilution and expanded. Hybridoma cells were injected intraperitoneally into BALB/c mice, primed with 2 ,6,10,14-tetramethyl pentadecane (pristene; Sigma, Sydney) to produce ascitic fluid.
25 Immunoassays: Screening for antibody production was achieved by a crude EIA. Flat bottom Maxisorp Immunoplates (Nunc) were coated overnight (12-18h), at 4 C, with heparin-binding proteins (37.5 mg PAPP-A/well; See Isolation 1) extracted from pooled pregnancy serum 38 weeks gestation: n 250).
The plates were washed once with 50 mM sodium phosphate buffer, pH 7.4, containing 0.15M NaCi (PBS). Unreacted protein binding sites were blocked, by incubating for min at room temperature (RT; 21-24 with PBS containing 0.1% bovine serum albumin (BSA) and 0.2% H 2 0 2 Plates were washed (X3) with PBS containing 0.1% BSA and 0.1% Tween-20 (T-PBS). Spent hybridoma culture media (250 jil) was incubated for 2hr at room temperature after 20 which the plates were washed (X3) with T-PBS and incubated, for 1h at room temperature, with horseradish peroxidase conjugated rabbit anti-mouse IgG (200 il/well; Dakopatts, Sydney), diluted 1/5000 in T-PBS. The plates were washed (X3) with T-PBS and incubated in the dark, for 15 min at RT, with substrate (200 pl/well; 0phenylenediamine (OPD), 0.67 mg/ml), in 0.1M citric acid phosphate buffer, pH 5.0, containing 0.012% H 2 0 2 The reaction was terminated by addition of IM sulphuric acid (100 pl/well) and absorbance measured at 490 nm on a microplate reader (Biotek).
Positive hybridoma media were screened by PAPP-A RIA. The final incubation volume (200 Al) consisted of hybridoma medium (100 il) and tracer 125 I-PAPP-A; 50 000 cpm/100 fl) After overnight incubation at room temperature, pooled serum (50 1l) obtained from normal human males was added to each tube and antibody bound tracer was precipitated by the second antibodypolyethylene glycol method (Sinosich et al., 1982) 9 The supernatant was aspirated and precipitated radioactivity measured by a multichannel gamma-counter (NE, Sydney).
Murine immunoglobulins were typed by EIA using a panel of rabbit anti-mouse immunoglobulins (IgG 1 IgG2a, IgG 2 b, IgG 3 IgM, IgA, K light Chain, K Chain; Biorad 25 Laboratories, Sydney).
A rabbit polyclonal antibody was prepared by repeated intramuscular injections of pure PAPP-A (100 After a positive test bleed was obtained, the animal was boosted with 100 pg PAPP-A administered subcutaneously. Antibody responses were monitored by RIA on blood samples obtained from the marginal ear vein.
Biotinylation of Immunoglobulins: Anti-PAPP-A IgG molecules were isolated from polyclonal and monoclonal antisera by affinity chromatography on a 3 ml column of Protein-A Sepharose (Pharmacia, Sydney), as per manufacturer's instructions.
Matrix bound proteins were desorbed with 0.2M glycine-HCl, pH 3.0, and dialysed (3 X 20 volumes) 21 against PBS. Approximately 1 mg of polyclonal IgG was recovered per ml of rabbit serum and 2.5 mg of IgG was recovered from 50 ml of culture supernate. Each preparation (1 mg/ml) was biotinylated with the long-chain derivative of N-hydroxysuccinimido-biotin (NHS-LC-biotin), as per manufacturer's instructions (Amersham, Sydney). After 1hr incubation at room temperature, unconjugated biotin was separated from the protein on a PD10 (Sephadex G-25M) column (Pharmacia) developed with PBS.
Development of PAPP-A Enzymeimmunoassay (EIA): Micro-ELISA (96 well) plates were coated, overnight at 4 C, with polyclonal rabbit anti-PAPP-A IgG in PBS.
After coating, the plates were washed with PBS and unreacted sites blocked (as detailed above). Assay reaction volume was 200 p.l with all samples/standards assayed in duplicate. Serum obtained from women (n>250) at advance stages of gestation 38 weeks) was pooled (LPS) and designated as 100 IU PAPP-A/L. The pooled LPS 20 was serially diluted (1/16-1/8196) to prepare a standard curve, 12.2-6246 mIU/L).
Affinity Immunoelectrophoresis (AIE): Pooled late pregnancy serum (38 weeks gestation; LPS) was incubated, overnight at room temperature, with media (control) or monoclonal antibody (5-62, 27-66, 1, 18-9) in a 1:1 ratio An aliquot (10 Al) was electrophoresed in the first dimension, in the absence and presence of heparin 20 units/ml gel), at 10 V/cm until the bromophenol blue marker migrated 3 cm. The gel was sliced and migrated in the second dimension into gel containing polyclonal rabbit anti-PAPP-A antibodies (Dakopatts) at 1/150 dilution. Second dimensional electrophoresis (at 2.5 V/cm) was performed overnight and the agarose gels processed for visualisation. Changes in migration distance (mm) were expressed as percentage of control value 27 S:22604E 10/10/95 22 Immunohistochemistry
(ICC):
Term human placenta, obtained by caesarean section, was sliced and fixed in phosphate buffered formalin pH 7.4, for 24h at 4 C. The tissue was then washed in phosphate buffered saline, pH 7.4, for 24h and processed by standard histological techniques. Sections (5 pm) were deparaffinised and processed for immunohistochemistry with polyclonal rabbit antichorionic gonadotrophin (Dakopatts), PAPP-A (Sinosich et al. 1987) and monoclonal anti-PAPP-A (clone 25-1).
Visualisation was achieved by enzyme bridge immunoperoxidase system using DAB as substrate. Negative controls included non-immune rabbit serum, mouse serum and culture media.
RESULTS
A total of five anti-PAPP-A clones were obtained; 1) clone 25-1, isotyped as IgG 1 with K chain, 2) clone 27-66, isotyped as IgM with K chain, 3) clone 62, isotyped as IgG 1 with K chain, 4) clone 18-9, 20 isotyped as IgG with K chain, and, 5) clone 41-1, which has yet to be characterised. By enzyme immunoassay, none of these antibodies reacted with immobilised heparin- Sepharose binding proteins extracted from normal male serum. The affinity constants (M- 1 for clones 25-1 and 5-62 were 1.26 x 10- 9 and 2.65 x 10- 9 respectively.
A sandwich enzyme immunoassay was developed using immobilised rabbit polyclonal anti-PAPP-A immunoglobulins as capture phase and clone 25-1 immunoglobulins for PAPP- A quantification (Fig This assay protocol is applicable to other PAPP-A monoclonal antibodies, but only clone 25-1 will be detailed.
Capture phase may include: 1) polyclonal PAPP-A antibody, 2) monoclonal PAPP-A antibody, 3) ligands, such as heparin, immobilised metal (zinc, copper, cobalt, nickel) cations and lectins (Con A, PHA) as detailed before, 4) specific, such as receptors, and non-specific, S:22604E 10/10/95 *t 23 such as dyes, ligands for PAPP-A.
Coating concentrations of rabbit immunoglobulins ranged from 0.38-6.0 Ag/well. At concentrations in excess of 1.5 gg/well, the increase in absorbance (at 490 nm) was insignificant. Thus, the EIA was developed on a coating concentration of 1.5 Ag rabbit immunoglobulins per well. (See Figure 4).
The influence of time and temperature on PAPP-A capture by immobilised polyclonal anti-PAPP-A immunoglobulins is shown in Fig 5. Independent of PAPP-A doses (0-6250 mIU/L) and temperature (4 0 C; room temperature, 19-23 0 C; 37 0 binding equilibrium was attained by the capture antibody within 24h. At room temperature or 37 0 C, maximal PAPP-A capture was achieved 15 after overnight incubation (15h) and these conditions (overnight incubation at room temperature) were selected for assay development.
Using biotinylated 25-1 antibody, at varied dilutions 1/50, 1/500, 1/5000), binding equilibrium was achieved (at room temperature) by 4h. (Fig. 6) The time required to attain equilibrium was inversely proportional to antibody concentration, with maximal binding being achieved within 30 min in excess antibody S" (1/5 dilution). Using these conditions (4h at room 25 temperature), Figure 7 shows typical PAPP-A dose-response curves at two dilutions (1/75, 1/150) of clone 25-1. For greater sensitivity the monoclonal antibody was diluted 1/75, but for routine assays not requiring enhanced sensitivity the antibody was diluted 1/150. Assay detection limit (n=20 replicates) was 21 mIU/L, with inter-assay precision of 6.2% and 10.5%. At PAPP-A doses of 100 and 1000 mIU/L and intra-assay precision was 9.2% (100 mIU/L) and 11.3% (1000 mIU/L), respectively. Total turnaround time for this assay was 24hrs.
Specificity was assessed in two ways. Firstly, by incubating each monoclonal PAPP-A antibody with immobilised heparin-Sepharose binding proteins, extracted from normal male serum. No significant reaction was f 24 detected. Secondly, at a fixed dose of PAPP-A (390.4 mIU/L) various monoclonal and polyclonal antisera (1/100 dilution) were tested for competition against biotinylated PAPP-A monoclonal 5-62, 25-1 (Table 1) and 18-9 (not shown). Positive reactions suppression in absorbance) were demonstrated with polyclonal antihuman, anti-monkey (rhesus) PAPP-A and rabbit anti-human placenta antisera (Table Positive controls included the same monoclonal antibody as the test, but in the nonbiotinylated form. All of the other antisera (PZP, a2M, SPI, complement factors 3 and 4, etc. Table 2) yielded negative results. Since the monoclonal anti-PAPP-A antibodies did not inhibit each other, except in a positive (self-self) format, these monoclonals recognise 15 distinct epitopes.
Serially diluted late pregnancy serum, and extracts of placentae, delivered either by caesarean section or spontaneously per vagina, were analysed by RIA and EIA using clone 25-1 as primary antibody. Slopes of the logit-log regressed lines (Fig. 8 9) suggests parallelism between circulating and tissue PAPP-A.
However, comparison of dose-response for extracts of human, chimpanzee and rhesus placentae showed marked species dependent responses. Clone 27-66 did not 25 distinguish between three primate PAPP-A species Clones 5-62 and 18-9 (not shown) demonstrated parallel lines only between human (m 1.82) and chimpanzee (m 2.13) PAPP-A (Fig. 11) In contrast, clone 25-1 reacted only with human (m 3.32), but not with chimpanzee (m 0.36) or rhesus (m 0.31) PAPP-A (Fig 12).
After incubation with a variety of glycosidases immunoreactivity of radiolabelled (1251-) PAPP-A to the two IgG clones (25-1, 5-62) was unchanged. Incubation with a-glucosidase and neuraminidase caused a marked increase in NSB, so that it was not possible to determine the true 'Bo By contrast, incubation of PAPP-A tracer with proteases (trypsin, elastase) markedly S:22604E 10/10/95 25 reduced PAPP-A immunoreactivity. These findings suggest the monoclonal antibodies recognise protide epitopes, rather than carbohydrate moieties.
By two dimensional immunoelectrophoresis, PAPP-A had a 2-electrophoretic mobility (39 mm). In the presence of IgG (5-62, 25-1, 18-9) and IgM (27-66) clones, PAPP-A mobility was reduced by 25.6% and 43.6%, respectively.
In the presence of heparin (20 units/ml gel), PAPP-A migration was increased by 33.3%. As none of the four clones inhibited the heparin-PAPP-A interaction, these epitopes are not associated with the heparin binding site of PAPP-A.
Immunohistochemical localisation of PAPP-A (clone 25-1) in human term placenta was limited to the syncytial layer. No PAPP-A immunoreactivity was detected in foetal or maternal blood vessels or villous stroma.
The monoclonal antibody is typically used for diagnostic or therapeutic purposes in unlabelled and/or labelled form. Typical labels include fluorescent, biotin, radioisotopic, chromogenic, chemiluminescent, electron dense, magnetic labels and enzyme labels, as examples. Labelling may also be achieved by means of a second antibody against the primary antibody with the second antibody being detectably labelled.
PAPP-A measurement in female (ovarian follicular fluid, cervical mucus) and male (seminal plasma, prostatic fluid) reproductive tract secretions can be of use to assess folliculogenesis, granulosa cell status, ovulation and pathology of the male accessory glands, such as prostate gland. Each fluid should be centrifuged to remove cells and the supernate stored at -20 0 C, until required for analysis. Cervical mucus should be extracted with 500 ul of assay buffer, centrifuged to remove cells and cellular debris, and supernate stored at -20 0 °C until required for analysis.
Blood, obtained by routine venepuncture, can be collected into anticoagulant free tubes or into anticoagulant (heparin, EDTA, citrate, as examples) S:22604E 10/10/95 v 26 containing tubes. In each case the sample should be centrifuged to separate the serum/plasma phase from blood cells. The supernate fluid is aspirated and stored at until required for analysis.
In the case where trophoblast cells are to be isolated, anticoagulated (EDTA, citrate, heparin) maternal blood or vaginal fluid is washed with physiological buffers or culture media to remove the plasma. The sedimented cells are resuspended in buffer or culture media and incubated with one or more PAPP-A polyclonal or monoclonal antibodies (or their derivatives). These PAPP-A antibodies may be free or immobilised, for example on magnetised particles. The free PAPP-A antibodies can be removed with a capture 15 phase, such as Protein-A Sepharose, secondary antibody immobilised on magnetic and non-magnetised particles.
The isolated trophoblast cells can be used for fetal karyotype, fluorescent in situ hybridisation (FISH), molecular biology, cell culture and immunocytochemical studies.
Tissue, for cell culture and immunohistochemical analyses, can include any desired normal and pathological tissue. Cell isolation and tissue processing was achieved by standard protocols as detailed above.
Immunodetection of PAPP-A, in maternal blood, can be used to assess pregnancy wellbeing and as part of a prenatal biochemical screen to assess fetal development.
In all adverse clinical situations, such as extra-uterine pregnancies, fetal death in utero, anembryonic pregnancy, incomplete abortion, spontaneous miscarriage, fetal malformation and/or aneuploidy (chromosomal abnormality), as examples, PAPP-A levels are depressed in maternal circulation. Although, in most of the pregnancy disorders, hCG (or free f3-subunit) expression correlates with PAPP-A levels. In Trisomy 21 (Down Syndrome) hCG (or free 0-subunit) expression is increased. Therefore, development and application of an algorithm which combines PAPP-A and hCG (or free f-subunit) measurements 1 *f 27 will permit discrimination of Trisomy 21 (Down Syndrome) from other pregnancy disorders. With a sensitive immunoassay, PAPP-A measurement can be established as an integral part of antenatal care in the first trimester of pregnancy.
In oncological situations, PAPP-A is expressed by tumor granulosa cells and gestational trophoblastic diseases (GTD) As the GTD becomes more aggressive and metastatic, PAPP-A- expression is downregulated. By contrast, hCG and free 0-subunit expression is upregulated. Again, application of a PAPP-A hCG (or free 3-subunit) algorithm enables discrimination between malignant and benign GTD.
e* INDUSTRIAL APPLICABILITY 15 In addition to the industrial applicability already indicated, it is envisaged that PAPP-A and PAPP-A variants can be used as a medicament to treat pregnant women exhibiting low levels of PAPP-A.
The amount of PAPP-A or variant required to produce a single dosage form will vary depending upon the condition to be treated, patient to be treated and the particular mode of administration. The specific dose level for any particular patient will depend upon a variety of factors including the activity of the molecule 25 employed, the age, body weight, general health and diet of the patient, time of administration, route of administration, rate of excretion, drug combination and the severity of the condition undergoing treatment.
.9 TABLE 1
-I
LIST OF ANTIBODIES CHECKED FOR CROSSREACTIVITY AGAINST THE PAPP-A MONOCLONALS DAKO ANTIBODIES alphafetoprotein, albumin, al-antichymotrypsin, antithrombin 111, alantitrypsin, C reactive protein, Cl esterase inhibitor, C3C complement, C3C(unspecific), C3d complement, C4 complement, cholinesterase, fibrinogen, fibronectin, human chorionic gonadotropin, human placental lactogen, inter-atrypsin inhibitor, B-lipoproptein, a2-macroglobulin orosomucoid, PAPP-A, a2plasmin inhibitor, plasminogen, pregnancy zone protein, schwangerschaftsprotein 1, placenta and B2-microglobulins.
PROTEIN A-ISOLATED RABBIT SERUM Pregnancy protein 12, pregnancy-associated plasma protein B, and chimp PAPP-A.
RABBIT SERUM Human serum PAPP-A 1&2, chimp PAPP-A, rhesus PAPP-A, heparin-bound macaque LPS, macaque male serum, heparin-bound human male serum, heparin bound guinea pig placental homogenate, guinea pig placental proteins 1&2, seminal plasma, pregnancy protein 10, pregnancy protein 14, pregnancy protein 5, and inhibin.
MONOCLONAL ANTIBODY SUPERNATANTS 25-1 IgG, 25-35 IgG, 1 27-66 IgM, 27-105 IgM, 1 5-62 IgG, 5-12 IgG, 1 5-39 IgG, 1 17 IgG, 1 11-1-30 IgM, 2 S:22604E 10/10/95 9 0 0 0 0 .09 00 *00 0 9 9* 0 0 0 9 0 9 9 *0 9 0 990 *0 9 0 00 0 00 0 *q9 TABLE 2 SPECIFICITY REDUCTION IN O.D.
25-1 5-62 Dako anti-PAPP-A -78.40- -91.90, Dako anti-Placenta -47.8%o -56.29g Rabbit anti-Human serum PAPP-A 1 -71.3% -96.1?6 Rabbit anti-Human serum PAPP-A 2 -72.6% -90.5% Rabbit anti-Rhesus PAPP-A -86.3%- Protein-A isolated anti-Chimp PAPP-A -60.4%6 -29.9%- 25-1 Monoclonal antibody supernatant -50.8-0 25-35 Monoclonal antibody supernatant -46.60% 5-62 Monoclonal antibody supernatant 61.70s 5-23 Monoclonal antibody supernatant 60.8%- 5-39 Monoclonal antibody supernatant -62.6%i 5-17 Monoclonal antibody supernatant 59.5%0 30 LIST OF REFERENCES 1. Lin, Halbert, and Kiefer, D.: Characterization and purification of human pregnancy associated plasma proteins. Fed. Proc. 32: 5232, 1973.
2. Sinosich, Teisner, Folkersen, et al.: Radioimmunoassay for pregnancy-associated plasma protein-A. Clin. Chem. 28: 50, 1982.
3. Wald, Stone, Cuckle, et al.: First 10 trimester concentrations of pregnancy associated plasma protein A and placental protein 14 in Down's syndrome; BMJ Vol. 305, p-28 (1992).
4. Westergaard, Chemnitz, Teisner, et al.: Pregnancy-associated plasma protein-A: A possible marker in the classification of prenatal diagnosis of Cornelia de Longe Syndrome.
Prenat.Diag 3:225, 1983.
Sinosich, Davey, Teisner, B. et al.: Comparative studies of pregnancy associated plasma 20 protein-A and 2 -macroglobulin using metal chelate chromatography. Biochem. Internatl. 7: 33, 1983.
S* 6. Sinosich, Porter, Sloss, et al.: Pregnancy associated plasma protein-A (PAPP-A) in human ovarian follicular fluid. J.Clin. Endocrinol.
Metab. 58: 500, 1984.
7. Sinosich, King, Bonifacio, et al.: Pregnancy associated plasma protein-A in human seminal plasma. Prot.Biol.Fluids 32:289, 1985.
8. Tsakok, Koh,S., Chua, et al.: Prognostic significance of the new placental proteins in trophoblastic disease. Br. J. Obstet.
Gynaecol. 90:483, 1983.
9. Davey, Teisner, Sinosich, et al: Interaction between heparin and human pegnancy associated plasma protein A: A simple purification procedure. Anal. Biochem. 131: 18, 1983.
31 Sinosich, Davey, Ghosh, et al.: Specific inhibition of human granulocyte elastase by human pregnancy-associated plasma protein-A.
Biochem. Internat. 5: 777, 1982.
11. Sinosich, M.J. and Saunders, Potential role for pregnancy-associated plasma protein-A in human reproduction. J. Reprod. Immunol. 10:55- 1987.
12. Sinosich, Saunders, D.M. and Grudzinskas, Pregnancy-associated plasma protein A: a barrier to maternal proteolytic attack In In Vitro Fertilization, Embryo Transfer and Early Pregnancy Edited by R.F. Harrison, J. Bonnar and W. Thompson, Lancaster, MTP Press Ltd 1984, pp.209.
13. Schindler, A.M. and Bischof, Histochemical 15 localization of pregnancy-associated plasma protein-A in fetal, infant and adult organs and comparison between antisera. Gynecol.
Obstet.Invest. 18:88, 1984.
0- 14. Bischof, Herrmann, W.L. and Sizonenko, P.C.: Pulsatile secretion of pregnancy-associated plasma protein-A (PAPP-A) in non-pregnant women: Br. J.
Obstet Gynecol 93:600, 1986.
Bischof, Reyes, Herrmann, W.L. et al.: Circulating levels of pregnancy-associated plasma 25 protein-A (PAPP-A) and human chorionic gonadotrophin (hCG) in intrauterine and extrauterine pregnancies.: Br. J. Obstet. Gynecol 90:323, 1983.
16. Sinosich M.J. Past, present and future of pregnancy associated plasma protein A In Placental and Endometrial Proteins. Tomoda, Mizutani, Norita, 0. and Klopper, A. (eds). USP, 1988, pp.11.
17. Sinosich, Smith, Grudzinskas, J.G. et al. The prediction of pregnancy failure by measurement of pregnancy-associated plasma protein-A (PAPP-A) followng IVF-ET. Fertil.Steril 40:539,1983.
18. Westergaard, Sinosich, Bugge, et 32 al. Pregnancy associated plasma protein A in the prediction of early pregnancy failure. Am.
J.
Obstet. Gynecol. 145: 67, 1983.
19. Sinosich, Ferrier, Saunders, D.M. S Monitoring of post-implantation embryo viability following successful in vitro fertilization and embryo transfer by measurement of- placental proteins. Fertil. Steril. 44: 70. 1985.
Sinosich, Ferrier, Porter, et al. Circulating and tissue concentrations of pregnancy associated plasma protein-A (PAPP-A) in tubal eptopic gestation. J. Clin. Reprod.Fertil u b a l 1985. Reprod.Fertil. _:3 S21. Brambati, Lanzani, Tului, et al. 15 Ultrasound and- e. Ultrasound and biochemical assessment of first trimester of pregnancy In The embryo: normal and abnormal development and growth: Chapman,
M.,
Verudzinska, chard, T. (eds) London, Springer- Verlag, pp. 181-94 (1990).
22. Lin et al. Characterisation of four human pregancy a associated plasma proteins Am. J. Obstet. Gynecl 18:223-26, 1974.
23. Lin, Holbert, S.P. and Kiefer
D
pregnancy associated human plasma proteins: purification, monospecific anti-sera and immunological identification Int. Archs Allergy Appl. Immun. 47: 35 (1974).
24. Bischof, P. Purification and characterization of pregnancy-associated plasma protein-A (PAPP-A): Arch. Gynecol. 227: 315-32 (1979) Sutcliffe, Kukulska, Nicholson,
L.V.B.,
et al. The use of antibody affinity chromatography and other methods in the study of pregnancyassociated proteins. In Placental Proteins (Klopper. A, and Chard eds. pp. 55-70, Springer- Verag Berlin/New York.) 26. Folkersen, Grudzinskas, Hindersson, et al. Purification of pregnancy-associated plasma 33 protein-A by a two step affinity chromatographic procedure (1981). Placenta 2: 11, (1981).
27. Sinosich, Teisner, Davey,M., etal.
Pregnancy-associated plasma protein-A: interaction with heparin in crossed affinity immunoelectrophoresis. Aust. N.Z. J. Med. 11: 429, (1981) 28. Sinosich, M.J. Biological role of pregnancyassociated plasma protein-A in human reproduction.
In Proteins of the Placenta Bischof, P and Klopper, A (eds). Kasper, Basel. pp. 158 (1985).
29. Sinosich, Sim, Teisner, B.
Characterization of pregnancy associated plasma protein-A: comparison with a 2-macroglobulin.
Biochem Inter 20: 579, (1990) Sinosich, M.J. Zakher, A. Pregnancy-associated plasma protein-A (PAPP-A) interaction with heparin; A critical appraisal. Gynecol Obstet Invest 32:72, (1991).
31. Maniatis et al. (1984): Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press.
25 In the claims which follow and in the preceding summary of the invention, except where the context *o requires otherwise due to express language or necessary implication, the word "comprising" is used in the sense of "including", i.e. the features specified may be associated with further features in various embodiments of the invention.
o It is to be understood that a reference herein to a prior art document does not constitute an admission that the document forms part of the common general knowledge in *9 35 the art in Australia or in any other country.
o
Claims (28)
1. A method for purifying PAPP-A from a sample comprising the steps of: Applying the sample to a positive affinity chromatography on Heparin-Sepharose and (ii) size fractionating the fractions obtained from step on a gel filtration column; and (iii) applying the fractions obtained from step (ii) to an anion-exchange column by HPLC; and (iv) applying the fractions obtained from step (iii) to negative affinity chromatography on a matrix with immobilised antibodies.
2. The method according to claim 1, wherein the steps (ii) and (iii) can be performed in any order.
3. The method according to claim 1, further comprising applying the fractions obtained from the matrix to a Heparin-Sepharose column.
4. PAPP-A protein purified by the method according to any one of claims 1, 2, or 3.
5. PAPP-A protein according to claim 4, which is a homotetramer having a molecular weight of approximately 820 kilo Daltons and an isoelectric point of approximately 4.5, each monomeric subunit of the homotetramer having a 30 molecular weight of approximately 200 kilo Daltons and an N-terminal amino-acid sequence of Glu-Ala-Arg-Gly-Ala-Pro- Glu-Glu-Pro-Ser-Pro-Pro-Ser.
6. A variant of the protein according to claims 4 or 5 which corresponds to or comprises a portion of PAPP-A or has homology with the PAPP-A amino acid sequence, and which has the biological or immunological activity of 9 S as .9 o0 a* S: P31771 35 PAPP-A.
7. A polynucleotide encoding PAPP-A according to claim 4 or a PAPP-A variant according to claim 6.
8. A monoclonal antibody which recognises PAPP-A but does not significantly cross-react with immobilised heparin-Sepharose binding proteins extracted from normal male serum.
9. A monoclonal antibody according to claim 8 which is an IgG or IgM antibody. A monoclonal antibody according to claim 8 which has kappa light chains.
11. A monoclonal antibody having an IgG 1 with K light chain isotype with an affinity constant of 1.26x10 9 which reacts specifically with human PAPP-A but not chimpanzee or rhesus PAPP-A.
12. A monoclonal antibody having an IgM with K light chain isotype which specifically recognises PAPP-A from human, chimpanzee and rhesus primates.
13. A monoclonal antibody having an IgG with K light chain isotype which reacts specifically with human and ;chimpanzee PAPP-A but not rhesus PAPP-A. t o
14. A monoclonal antibody having an IgGi with K light chain isotype with an affinity constant of 2.65xl0 9 which reacts specifically with human and chimpanzee PAPP-A but not with rhesus PAPP-A. S* e
15. A monoclonal antibody produced by clones 25-1 or
18-9, as herein before defined, or an antibody or fragment thereof, which recognises the same epitope as an antibody 0 itC S: P31771 36 produced by one of the clones. 16. A monoclonal antibody according to any one of claims 8 to 15 wherein the PAPP-A used to generate the antibody is at least 90% pure. 17. An assay for PAPP-A comprising using a monoclonal antibody according to any one of claims 8 to 16 to detect PAPP-A. 18. An assay according to claim 17 which is an ELISA assay, immunoradiometric assay or any other format.
19. An assay according to claim 17 comprising a specific or a non-specific ligand as capture phase. An assay according to claim 17 wherein the monoclonal antibody may or may not be directly tagged.
21. A method of detecting PAPP-A which comprises the steps of: contacting the sample with a PAPP-A capture phase which consists of one or more of the following: monoclonal PAPP-A antibody; 25 (ii) polyclonal PAPP-A antibody; (iii) immobilised heparin; (iv) immobilised divalent metal cations; immobilised lectins; and (vi) other specific receptors and non-specific dye ligands with which PAPP-A has an affinity; contacting the immobilised or captured PAPP-A sample with a monoclonal antibody according to any one of claims 8 to 16 labelled with a detectable marker; incubating the sample and the labelled antibody 35 to permit the labelled antibody to bind to any PAPP-A in the sample; and detecting the labelled portion. S: P31771 37
22. A method according to claim 21 wherein the immobilised divalent cations are selected from Cu 2 Zn 2 Co 2 and Ni 2
23. A method according to claim 21 wherein the immobilised lectin is selected from Concanavalin A, Helix pomatia, Lens culinaris, Limulus polyphemus, phytohaemmaglutinin, Ricnus communis and wheat germ lectin, or a lectin with an equivalent to one of these lectins.
24. A kit for detection of PAPP-A in a sample, said kit comprising at least one monoclonal antibody according to any one of claims 8 to 16 together with a positive control or a negative control. PAPP-A according to any one of claims 4 to 6 for use in a method of treatment of a pregnant pati nt exhibiting the absence or low levels of PAPP-'A
26. A medicament comprising an effective amount of PAPP-A according to any one of claims 4 to 6 together with a pharmaceutically acceptable carrier, diluent or 25 excipient.
27. Use of PAPP-A according to any one of claims 4 to 6 as a contraceptive agent. 30
28. PAPP-A monoclonal antibodies according to any o*oa one of claims 8 to 16 for use in a method of treatment of unwanted conceptions, abnormal pregnancies and/or trophoblastic tumours. 35
29. Use of PAPP-A monoclonal antibodies according to any one of claims 8 to 16 for the isolation of trophoblast cells, from maternal circulation or reproductive tract, P31771 38 for prenatal fetal karyoptyping. Use of PAPP-A monoclonal antibodies according to any one of claim 8 to 16 for the diagnosis of oncological disorders and compromised pregnancies.
31. Use of PAPP-A monoclonal antibodies according to any one of claims 8 to 16 for prenatal screening for Downs Syndrome and diagnosis of malignant tropoblastic disease.
32. An epitope of PAPP-A, being an epitope recognised by at least one of the monoclonal antibodies of claims 8 to 16.
33. The protein PAPP-A substantially free of other primate proteins as herein before described with reference to the accompanying Examples.
34. A monoclonal antibody which recognises PAPP-A substantially as herein described with reference to the accompanying Examples. *e Dated this 2 8 th day of September 2000 S. 25 NORTHERN SYDNEY AREA HEALTH SEARVICE AND MICHAEL JOSEPH SINOSICH By their Patent Attorneys S" GRIFFITH HACK A o o p :P31771
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU78548/98A AU726994B2 (en) | 1993-03-19 | 1998-07-23 | PAPP-A, its immunodetection and uses |
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| AUPL7926 | 1993-03-19 | ||
| AU63701/94A AU6370194A (en) | 1993-03-19 | 1994-03-21 | Papp-a, its immunodetection and uses |
| AU78548/98A AU726994B2 (en) | 1993-03-19 | 1998-07-23 | PAPP-A, its immunodetection and uses |
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| AU63701/94A Division AU6370194A (en) | 1993-03-19 | 1994-03-21 | Papp-a, its immunodetection and uses |
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| AU726994B2 true AU726994B2 (en) | 2000-11-30 |
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| AU78548/98A Expired AU726994B2 (en) | 1993-03-19 | 1998-07-23 | PAPP-A, its immunodetection and uses |
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| AU (1) | AU726994B2 (en) |
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1998
- 1998-07-23 AU AU78548/98A patent/AU726994B2/en not_active Expired
Non-Patent Citations (3)
| Title |
|---|
| E.A. MOWLES ET AL. J.IMMUNOLOGICAL METH., 95, 129-131 (1986) * |
| M.J. SINOSICH ET AL. PLACENTA 10, 569-578 (1989) * |
| MW.DAVEY ET AL. ANALYTICAL BIOCHEMISTRY 131, 18-24 (1983) * |
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