MONOCLONAL ANTIBODIES SPECIFIC FOR ACETYL CHOLINESTERASE

Abstract: A monoclonal antibody which is specific for human secretory acetylcholinesterase (sAChE) and which shows no cross-reactivity with other types of human acetyl cholinesterase is useful for determining human sAChE in a sample and consequently for detecting Neural Tube Defects such as anencephaly, encephalocele and spina bifida.

Description: MONOCLONAL ANTIBODIES SPECIFIC FOR ACETYL CHOLINESTERASE This invention relates to monoclonal antibodies, to their preparation and to their use in immunoasεayε. Neural Tube Defects (NTDs) are a number of structural anomalies of the central nervous system (CNS) of the human fetus. They result from the failure of the neural tube to close properly during embryogenesiε. There are three major types of NTD: (1) Anencephaly: where the closure defect can occur at the top of the neural tube. It results in babies being stillborn. (2) Encephalocele: is relatively uncommon, with the closure defect occurring a little lower down the neuroaxiε. Babies do survive with little paralysis, and they usually grow up spastic, severely retarded and often blind. (3) Spina bifida: where the closure defect occurs lower still resulting in babies being born with a portion of the spinal cord exposed. This results in paralysis of the legs, incontinence and hip, knee and feet deformities. About 90% of these babies will die if not operated on. NTD incidence varies considerably throughout the world. In Europe, the prevalence is 6-13 per 10,000 births. The risk goes up considerably in women over 35 years of age. Currently NTD'ε are diagnosed by: (1) Genetic counselling: by checking the family history to find out if a couple is in a risk group, i.e. if they have a previously affected child or parent or close relative. A drawback of this method is that over 95% of all NTDs occur in couples not previously considered at risk from this disorder. (2) Ultra-sound scanning (US) : has proved very useful in identifying many fetal abnormalities. However, there are some drawbacks: (a) US has 100% accuracy in detecting anencephaly but only 80% for spina bifida. (b) It relies on the experience of the operator of the scanner. The risk of missing malformation is estimated at 1.6%. (c) Misdiagnoεis occurs if scanning is done at a very early gestational age. (3) Alpha-fetoprotein (AFP): is a glycoprotein with a molecular weight of 70,000 Daltonε. It iε uεually secreted by the embryo yolk sac and later by the fetal liver. It diffuses into the Amniotic Fluid (AF) and then into the maternal circulation. AFP levels were found to be elevated in maternal serum of women with NTD fetuseε. Currently, its measurement by an immunoaεsay iε the main diagnostic test, in conjunction with US, for NTD. Normally, AFP iε meaεured in maternal serum. An elevated value is followed by US scanning, then by amniocenteεiε. The amniotic fluid (AF) iε then teεted for AFP. However, there are problemε with AFP: (a) It requireε a preciεe knowledge of gestational age since the normal levels of AFP vary in maternal serum considerably with gestational age. (b) AFP levels in AF also vary with gestational age. Furthermore, a major problem with asεaying AFP in AF iε the likelihood of a falεe positive due to contamination of the AF samples with fetal blood and/or maternal blood. A U.K. collaborative study has found that only 80% of NTD are detected by AFP measurements. (4) Neural-specific acetyl cholinesterase (AChE) : since the main defect in NTD is the incomplete closure of the neural tube, many of the neurally-derived proteins and other co ponentε are expected to be found in the AF and to diffuεe into maternal serum. These could, therefore, be potential markers for NTD. In 1979, Smith et a_l. (Lancet, i, 685, 1979) proposed the analysis of AChE in AF as a supplementary test to improve the diagnosis of NTD. Cholinesterases are a group of enzymes that hydrolyse cholineεterε into choline. Pεeudo-cholineεteraεeε, which are preεent in serum, muscle and brain, have a substrate preference to butyryl cholines. Acetyl cholineεterases (AChE) have a substrate preference to acetyl cholines. The two types are different immunologically and biochemically. AChEs are of multiple forms, globular and asymmetric forms. The latter are found predominantly in muscle tissue. Globular forms are dominant in the central nervous system and red blood cells (RBC) . They exist as a monomer, dimer, or tetra er. Globular AChEs are membrane-bound or soluble. In human brain, about 70% of AChE iε membrane-bound and 100% of red blood cells AChE is membrane-bound. The central nervous system soluble AChE iε in itself of multi- forms. The bulk of it is believed to be derived from the membrane-bound AChE as iε εhown by its behaviour in the presence of detergentε. One type of the neural soluble AChE iε known aε the secretory AChE (sAChE) . It iε εecreted by neural cells, has a distinct electrophoretic mobility, is the only form of AChE found in mammalian cerebrospinal fluid (CSF) , is not influenced by detergentε and is the only form that binds selectively to the AChE inhibitor edrophoniu chloride when this iε immobiliεed on Sepharoεe (Trade Mark)-4B gel. The iεolation and partial characterisation of the secretory form of human brain AChE has been described (Gardner et a].., Biochem.Soc.Trans. , 14 ,1234- 1235,1986) . In amniotic fluid (AF) , there are three forms of AChE present in addition to pseudo-ChE. A monomer (4.0 S) , a dimer (5.5 S) , and a tetramer (10.3 S) . The origin of the first two iε not clear. However, the dimer iε very much εimilar to the dimer membrane-bound AChE found in red blood cellε (RBC) . The tetramer form iε the neural-specific form, the secretory AChE (sAChE) . The amount of this form goes up to 62-fold in NTD. AChE is detected in AF in three ways. These are: (a) Enzymatically: by using the standard Ellman test for assaying esteraseε. The test requires the uεe of highly specific inhibitors in order to be able to distinguish between AChE and pseudo-ChE. It can not distinguish between the different forms of AChE. Hence, it iε uεeless in caεeε where the AF samples are contaminated by fetal or maternal blood. A recent modification to this test involves a prior separation of the different molecular forms by sucrose density gradient centrifugation followed by the Ellman test. This makes the test complicated requiring ultra- centrifuges, and overnight spins. Furthermore, it does not distinguish between the sAChE and other brain-derived AChE with similar molecular weight. (b) Gel electrophoresiε and hiεtochemical staining: this iε the εtandard procedure currently employed in clinical laboratorieε. It relieε on the electrophoretic mobility of the sAChE. AF samples are electrophoresed on 6% acrylamide gels which are subsequently stained for esteraεe activity. Specific AChE activity is identified by using a εpecific inhibitor. A U.K. collaborative study has shown this test to be very uεeful, finding that AChE levelε do not vary with gestational age as in the case with AFP. Therefore, precise knowledge of fetal age iε not neceεεary. There are a number of drawbackε with this procedure, though: (i) It iε a qualitative test. (ii) The test is useless if AF samples are contaminated with fetal or maternal blood because serum pseudo-ChE and RBC AChE mask the specific sAChE band on the gel. (iii) The test is time-consuming as it requires electro- phoresis and histochemical staining. (c) Im unological testing: many different antibodies, both polyclonal and monoclonal, have been raised against different forms of AChE. Many of theεe do not cross react with pseudo-ChE. Zaneta et a_l (FEBS Lett.129,293,1981) raised a rabbit antiserum against rat brain membrane AChE, which had only low cross reactivity with soluble AChE. Gennari and Brodbeck (J. Neurochem. 11,697,1985) reported an antiserum raised to detergent soluble AChE from human brain which showed a moderate preference for detergent soluble versus the water soluble enzyme. An antibody raised to rabbit AChE binds the membrane-bound form of brain AChE better than the soluble form (Rakonczay and Brimijoin, BBA 832. 127,1985). Immunological differences between AChEs of different tissues are also coming to light. Most antibodies raised to AChE εhow equivalent affinity for brain and erythrocyte enzymes from the same species. Rasmusεen et al (Clin.Chim. Acta, 166,17,1987) evaluated 11 monoclonal antibodies raised to human RBC AChE with respect to reactivity with AChE from both RBC and brain. They claimed that one of their onoclonalε actually showed preference for membrane-bound AChE purified from human brain, but all their monoclonal antibodies reacted with RBC AChE. There are few reportε on the uεe of immunoaεεayε of AChE in the diagnoεiε of NTD. The main problemε are the difficulty of obtaining specificity to the neural and more exactly, the secretory AChE. Reported assays rely on the use of either monoclonal or polyclonal antisera raised to human brain AChE. The assays are of two main types„ In the first type of assay, the antigen, AChE, is sandwiched between two monoclonal or one monoclonal and one polyclonal antibodies (Brimijoin et al., J. Neurochem.49. 555,1987). The second type is also of the antigen-capture type but relies on the AChE's own enzyme activity. Monoclonal anti-RBC AChE antibodies are immobilised followed by incubation with test sample. Bound AChE is detected by using the Ellman test (Norgaard et a_l, Chin.Chim.,29.,1061,1983) . This second, approach does not require pure AChE. Varying degree of succesε have been reported for theεe aεεayε when uεed to diagnoεe NTD (Norgaard et a .,1983; Brock and Bader,Clin.Chim.Acta 127, 419,1983; Brimijoin et al, Fed.Proc.16,2557,1987; Brock et al. , Lancet i,5,1985; Sorensen et al,. , Prenat.Diag Z,75, 1987) . There are a number of problems with immunoaεεayε of AChE in the diagnoεiε of NTD: (i) Scarcity of material has meant difficulty in raising antisera to neural AChE. (ii) Currently available antibodieε are raiεed to RBC AChE and membrane-bound AChE. They are uεeleεs in detecting AChE in AF if the AF iε contaminated with fetal or maternal blood, due to the relatively huge amountε of RBC AChE preεent in εuch samples. (iii) Although it iε not entirely clear, all the current antisera are thought to react with many types of AChE. They are not specific for the sAChE or at least to the brain-derived AChE. Measurement of AChE in maternal serum has often been proposed as a teεt for the prenatal diagnoεiε of NTDε. Such a test would make it possible.to avoid doing amnio- centeεiε with all the riεkε to the fetuε involved in thiε procedure. However, such a teεt iε not possible yet because of the presence of pseudo-ChE and a number of AChE forms in the serum, as already mentioned. Fetal and adult human sAChE have now been purified. Using these and special techniques, immortalised cell lines have been obtained which secrete monoclonal antibody specific for human sAChE. Standard jln vivo immunisation methods of preparing hybridomaε did not result in cell lines which secrete human sAChE-specific antibody. Specific screening strategies were adopted in order to detect hybridomas producing the monoclonal antibody εpecific for human εACheE. The monoclonal antibody can be uεed in immunoaεsays for fetal and adult human εAChE, thuε enabling NTDε to be detected. Accordingly, the present invention provides a monoclonal antibody which is specific for human sAChE and which shows no crosε-reactivity with other typeε of human acetylcholinesterase. The monoclonal antibody may be IgG or IgM. It may be rat,mouse or human monoclonal antibody-. The monoclonal antibody is prepared by a process which compriseε: (a) culturing an immortaliεed cell line which εecreteε the monoclonal antibody; and (b) isolating the monoclonal antibody thus produced. The invention also provides an immortalised cell line which secretes *a monoclonal antibody εpecific for human εAChE. Such a cell line can be prepared by one of four proceεεeε: Proceεε (I) compriεeε: (a) immunising an animal with adult or fetal human sAChE; (b) obtaining lymphatic tisεue cellε from the immuniεed animal ; (c) culturing the obtained cellε with adult or fetal human sAChE; (d) immortalising the cultured cells; and (e) screening the resulting cell lines for an immortaliεed cell line which secretes monoclonal antibody which is εpecific for human sAChE. Procesε (II) comprises: (a) obtaining lymphatic tisεue cells from an animal, (b) culturing the obtained cells with adult or fetal human sAChE, (c) immortalising the cultured cellε, and (d) screening the resulting cell lines for an immortalised cell line which secretes monoclonal antibody which is εpecific for human sAChE. Proceεε (III) compriεeε: (a) obtaining lymphatic tissue cellε from an animal, (b) culturing the obtained cells with adult or fetal human sAChE, (c) culturing the cells obtained in step (b) with freεh adult or fetal human sAChE, (d) immortalising the cells obtained in step (c) , and (e) screening the resulting cell lines for an immortaliεed cell line which secreteε monoclonal antibody which iε εpecific for human sAChE. Process (IV) comprises: (a) immunising an animal with adult or fetal human sAChE by inoculation directly into a lymphatic organ or lymph .node, (b) obtaining lymphatic tiεεue cells from the immunised animal, (c) immortalising the cells obtained, and (d) screening the resulting cell lines for an immortalised cell line which secretes monoclonal antibody which iε specific for human sAChE. Typically, the same type of human sAChE, adult or fetal, iε uεed in both εtep (a) and step (c) of procesε (I) and in both εtep (b) and step (c) of proceεε (III) . In one embodiment, εplenocyteε are obtained in step (b) of process (I) or (IV) or in step (a) of procesε (II) or (III) . Preferably the cultured cells are fused with myeloma cells in step (d) of process (I) or (III) or in step (c) of process (II) or (IV) . Generally resulting cell lines are screened by - contacting a solid support coated with adult or fetal human sAChE with supernatant from each cell line culture and with, in the presence of polyethylene glycol, a labelled antibody capable of binding to any monoclonal antibody in the supernatant which binds to the human sAChE; or - contacting a solid support coated with adult or fetal human sAChE with supernatant from a culture of each cell line grown in serum-free or low serum-supplemented medium and with a labelled antibody capable of binding to any monoclonal antibody in the supernatant which binds to the human sAChE. The invention further provideε a method of determining human εAChE in a sample, which method compriseε carrying out a εaid determination uεing a monoclonal antibody which iε εpecific for human εAChE and which shows no crosε- reactivity with other typeε of human acetylcholineεteraεe. The method may compriεe contacting a sample suspected of containing fetal human sAChE with a said monoclonal antibody and determining whether the said monoclonal antibody haε bound to any fetal human sAChE. The invention additional provideε a test kit suitable for use in determining fetal human εAChE, which kit compriεeε a monoclonal antibody which iε specific for fetal human sAChE and which shows no crosε-reactivity with other typeε of human acetylcholineεteraεe and means for determining whether the monoclonal antibody haε, in use, bound to human sAChE. The monoclonal antibodieε are specific for human sAChE. They react specifically with adult or fetal human sAChE and show no crosε-reactivity with other types of human acetylcholinesterase. In particular, they show no reactivity with human red blood cell AChE, and preferably no crosε-reactivity with human serum AChE, human me brane- bound neuronal AChE, human muscle AChE and neuronal-soluble non-secretory AChE. The antibodies do not croεε-react either with human serum pseudo-ChE. If raised to fetal sAChE, the monoclonal antibodies can bind preferentially to fetal human sAChE rather than to adult human sAChE. Immortalised cell lines which secrete such monoclonal antibodies require purified human sAChE for their preparation. This purified material iε obtained from human brainε. Fetal human brainε are typically obtained from aborted fetuεeε over 16 weekε old. The brainε are typically stored frozen, thawed and homogenised in ice-cold 0.3M sucroεe containing 2mM ethylene dia ine tetraacetic acid (EDTA) or uεing phoεphate buffered saline/2mM EDTA as the buffer. The homogenate may be subjected to one or more cycles of freezing and thawing, and then centrifuged. The supernatant is removed and optionally iε εubjected to gel filtration. Sephacryl S-200 can be used for this purpose. Fractions are collected having a protein content aε determined by optical denεity at 280 nm, and AChE activity aε determined by the Ellman aεsay. The εAChE in the εupernatant from centrifugation or in the fractionε collected during gel filtration iε then purified by affinity chro atography. A column of edrophonium chloride-epoxy-Sepharoεe gel may be uεed. Bound material iε eluted, from which edrophonium chloride is removed by gel filtration. Fractionε containing AChE activity are pooled and lyophiliεed. They can be reconεtituted in phoεphate-buffered εaline and frozen for storage. In thiε way human sAChE having an activity of 900-1500 unitε/mg may be obtained. An immortalised cell line may be prepared by any one of procesε (I) to (IV) . Prior to immortalisation: Procesε (I) . A non-human mammalian hoεt such as a mouse or rat Ls inoculated with the adult or fetal human sAChE. After a sufficient time has elapsed to enable the host to mount an antibody response to the protein, antibody- producing cells are removed from the host. Cells of lymphoid origin such as splenocytes, lymph node cellε or other lymphatic tiεsue or peripheral blood lymphocytes may be obtained from the immunised host. Procesε (II) . Cellε of lymphoid origin such as splenocytes, lymph node cellε or other lymphatic tissue or peripheral blend lymphocytes may be obtained from a mammalian host such aε a ouεe or rat. The cellε are incubated in vitro in a culture medium containing adult or fetal human sAChE. Typically 5 x 10 6 cells/ml are incubated with 0.8 μg/ml of sAChE in 15ml of culture medium in a 25 cm 2 flask. The culture medium may also contain thymocyte-conditioned medium, culture supernatant from EL4 cells, phorbol myristate acetate or other growth factors. Typically the incubation lastε for a minimum of 3 dayε, for example 4-10 dayε. A suitable period may be 5 days or 6 days. By increasing the incubation time up to 10 to 14 dayε in the presence of adjuvant peptides, a class switch from lgM to lgG antibody can be induced. The cultured cells are then immortalised. Procesε (III) . Cells of lymphoid origin such aε εplenocyteε lymph node cellε or other lymphatic tissue or peripheral blood lymphocytes may be obtained from a mammalian hoεt such aε a mouεe or rat. The cellε are incubated in accordance with the procedure for proceεε (II) . An in vitro booεt may be given by tranεferring the cellε to freεh culture medium containing sAChE. The culture medium may also contain thymocyte-conditioned medium, culture εupernatant from EL4 cells, phorbol myristate acetate or other growth factors. Typically this second incubation or any subsequent incubation lasts for a period from several hours to 10 days. A suitable period may be 5 days or 6 days. By increasing the incubation time up to 10 to 14 days in the presence of adjuvant peptides, a class switch from lgM to lgG antibody may be induced. The culture cells are then immortalised. Procesε (IV) . A non-human mammalian host such aε a mouεe or rat iε immunised with adult or fetal human sAChE by direct inoculation into a lymphatic organ or lymph node. An adjuvant may also be included in the inoculation. Typically a mouse iε anaeεthetiεed with hypnorm/midazolan. The fur iε clipped over the spleen and an incision 1-1.5 cm long is made just below the left set of ribs. The muεcle is separated using blunt dissection, and the lower pole of the spleen exteriorized through a small incision in the peritoneum. The sAChE iε inoculated directly into the εpleen and the εkin in εtitched. After a sufficient time has elapsed to enable the host to mount an antibody reεponεe to the protein, antibody-producing cells are removed from the host. Cellε of lymphoid origin εuch as splenocytes, lymph node cellε or other lymphatic tissue or peripheral blend lymphocytes may be obtained from the immunised host. Typically the lymphoid cellε removed are splenocytes, from a mouse, between 2-4 dayε following immuniεation. A εuitable period may be 3-5 dayε. The lymphoid cells are then immortalised. The cellε from the i muniεed hoεt are incubated in vitro in a culture medium containing further adult or fetal human sAChE. The culture medium may contain also thymocyte- conditioned medium or other cell growth factors. Typically the incubation lasts for a minimum of 3 days, for example from 4 to 6 days. A suitable period is 5 days. By increasing the incubation time up to 10 to 14 dayε in the preεence of adjuvant peptideε, a claεε switch from IgM to IgG antibody can be induced. For cells to be immortalised, they may be fused with cells of an immortalising cell line. This cell line may be a myeloma cell line, e.g. of a mouεe. or rat. Alternatively, or additionally, the cultured cells may be transformed with, for example, a virus. The resulting fusions are screened. An immortalised cell line, such as a hybridoma, secreting antibody specific for human sAChE is thereby selected. The fusionε are typically screened by an enzyme-linked immunosorbent assay (ELISA) . A solid support, such as microtitre wellε, iε coated with adult or fetal human εAChE. Supernatant from a culture of a fuεion iε incubated with the coated support. A labelled antibody capable of binding to any monoclonal antibody in the supernatant which binds to the human sAChE is added. The labelled antibody iε generally added after the coated support haε been incubated with the culture supernatant and the support has been washed. In one embodiment the labelled antibody iε incubated with the εupport in the preεence of a polyethylene glycol (PEG) . PEG 4000 or 6000 may be added. The amount of PEG - may be from 1 to 10% w/v, for example from 2 to 6% w/v and preferably about 4% w/v. In another embodiment, the labelled antibody iε incubated with the support in the absence of a PEG provided the εupernatant iε a supernatant from a cell line which has been grown in, or pulsed with, a serum-free medium or a medium containing 1% v/v or less of serum, typically fetal calf serum. The labelled antibody binds to any monoclonal antibody, originating from the culture εupernatant, which haε bound to the human sAChE coating the solid support. The label is typically an enzyme label such aε horse radish peroxidase. In this way fusions secreting antibody specific for human sAChE can be detected, after addition of a substrate for the enzyme in the case where an enzyme label is employed. The resulting immortalised cell line can be used to obtain monoclonal antibody specific for human sAChE. Monoclonal antibody is prepared by: (a) culturing an immortalised cell line which secretes the monoclonal antibody; and (b) isolating the antibody thus produced. Progeny of clones producing the desired antibody can be grown. Step (a) may be conducted in vitro in suitable culture media in tissue culture flasks, in a hollow fibre tisεue culture device or in a cell fermenter for example. Alternatively cells may be cultured in step (a) in vivo. They may be grown in vivo in laboratory animals, for example in laboratory animals, such as mice and rats. Cellε of the immortaliεed cell line may be implanted in a body cavity, such aε the abdominal cavity, of the animal and allowed to grow. The reεulting monoclonal antibody can be separated from the culture medium or from the body cavity fluid such as the aεciteε fluid of the animal by techniqueε εuch aε ammonium sulphate precipitation, ion exchange chromatography, gel filtration, affinity chromatography, high-performance liquid chromatography, etc. The monoclonal antibody can be used in the diagnosiε of NTDε and, in particular, anencephaly, encephalocele and spina bifida. The monoclonal antibody can also be uεed in the di ' agnoεiε of omphalocele, gaεtroεchiεiε, intrauterine death, eεophageal atresia, twin pregnancy with acardiac fetus, normal infant, teratoma, asciteε, cyεtic hygroma, hypoplaεia °f heart and lungε, cloacal exεtrophy, hydrocele, epidermolyεiε bulloεa dyεtrophica, and aplaεia cutiε congenita; aε well as other neurodegenerative disorderε εuch aε senile dementia, Parkinson'ε disease, and Alzheimer's disease. In each case, diagnosiε dependε upon detecting and meaεuring levelε of sAChE. For the diagnosiε of a pre-natal condition preferably a monoclonal antibody iε employed which has been raised using fetal sAChE. A monoclonal antibody which binds preferentially to fetal human sA ChE rather than to adult human sAChE can be employed. Adult or fetal human sAChE can therefore be assayed using the monoclonal antibody. A sample suspected of containing human sAChE is contacted with the monoclonal antibody. The sample may be a biological fluid such as amniotic fluid or maternal serum from a pregnant woman. An asεay may be performed qualitatively, semi-quantitatively or quantitatively. A variety of aεεay formatε may be employed. The monoclonal antibody can be used to capture adult or fetal human sAChE selectively onto a solid surface from solution, to label selectively thiε protein or both to capture and to label thiε protein. The antibody also may be used in a variety of homogeneous assay formatε in which the protein iε detected in solution with no separation of phases. The types of assay in which antibody is used to capture the protein from solution involve immobilization of the antibody onto a solid surface. Thiε εurface should be capable of being washed. The typeε of surfaces which may be used include polymerε of variouε typeε (moulded into icrotitre wellε; beads; dipstickε; aεpiration tipε; electrodes; and optical deviceε) , particleε (for example latex; stabilized red blood cells (erythrocyteε) ; bacterial or fungal cellε; spores; gold or other metallic or metal- containing εolε; organic εolε; and proteinaceouε colloidε; with the uεual size of the particle being from 0.005 to 5, for example from 0.1 to 5, microns), membranes (for example of nitrocellulose; paper; cellulose acetate; chemically- activated membranes such as Millipore Immobilon (Trade Mark) or Pall Biodyne (Trade Mark) ; and high porosity/high surface area membranes of an organic or inorganic material) . The attachment of the antibody to the surfaces can be by passive adsorption from a solution of optimum composition which may include surfactants, solvents, salts and/or chaotropes; or by active chemical bonding. Active bonding may be through a variety of reactive or activatable functional groups which may be exposed on the surface (for example condensing agents; active esters; acid halides; anhydrideε; amino, hydroxyl, or carboxyl groupε; sulphydryl groups; carbonyl groupε; diazo groupε; unεaturated groups). Immobilisation may be through a preliminary coating with protein A or antibody to murine immunoglobulin. After contacting (reacting) the εurface bearing the antibody with a teεt sample, allowing time for reaction and, where necesεary, separating or removing the excesε of. the εa ple by any of a variety of meanε (waεhing, centrifugation, filtration, application of a magnetic field, capillary action) , the captured protein is detected by any means ' which will give a detectable signal. For example, this may be achieved by use of a labelled molecule, in particular antibody, or particle aε deεcribed • above which will react with the capture protein. Alternatively, the activity of the immobiliεed AChE can be aεεayed. The detectable signal may be optical or radio-active or physico-chemical and may be provided either directly by labelling the molecule or particle, especially antibody, referred to with for example a dye, radiolabel, electroactive species, magnetically resonant specieε or fluorophore, or indirectly by labelling the molecule or particle with an enzyme itεelf capable of giving riεe to a measurable change of any εort. Alternatively the detectable εignal may be due to, for example, agglutination, diffraction effect or birefringent effect occurring if any of the εurfaceε referred to iε in the form of particleε. A preferred assay format is the sandwich assay. A firεt antibody capable of binding to fetal human sAChE captureε the protein onto a solid surface and a second antibody capable of binding to the protein labels the protein. At least one of the first antibody and the second antibody iε a monoclonal antibody of the invention. One of the antibodies, for example the first antibody, may be a polyclonal antibody. The capturing and labelling operations may be performed in any order or simultaneously. Typically the second antibody iε labelled with an enzyme εuch aε an alkaline phosphataεe or peroxidaεe. A uεeful εandwich aεεay therefore involveε contacting a teεt εample with enzyme-labelled monoclonal antibody, capturing the reεulting immune complex onto a solid surface using a monoclonal or polyclonal antibody capable of binding to human sAChE, removing any excesε labelled antibody and adding a εubstrate for the enzyme. The presence of the human sAChE sample is thuε revealed. Other formatε which may be employed are any of thoεe εuitable for immunoaεsayε including (1) agglutination with the monoclonal antibody adεorbed onto particleε of, for example, polyεtyrene latex; (2) an enzyme-linked immuno- adεorbant aεεay (ELISA) carried out in microtitre plateε, (3) a dipstick ELISA with an antibody-coated dipεtick, and (4) εandwich aεεayε uεing εmall magnetic particleε coated with capture antibody together with the monoclonal antibody labelled either with coloured particleε, or particleε with the potential of colour development, or with an enzyme or a fluorescent moiety. Test kits εuitable for use in determining human sAChE in a εample compriεe a monoclonal antibody according to the invention and meanε for determining whether the antibody binds to human εAChE. Specific components of the kits may be aε described above. The kits may also comprise one or more additional components selected from a control, buffer and diluent. A kit for use in an enzyme-immunoaεεay typically includeε an enzyme-labelled reagent and a substrate for the enzyme. The enzyme may either be bound to the monoclonal antibody of the invention which can bind to the protein of the invention or be bound to polyclonal or monoclonal antibody capable of binding to the monoclonal antibody. The following Examples illustrate the invention. A Reference Example iε alεo provided. In the accompanying drawingε: Figure 1 shows the effect of including PEG in the second step of a standard ELISA. Microwell plates coated with fetal sAChE (FsAChE) (200 ng/ml, lOOμl) , were incubated with dilutions of anti-sAChE culture supernatantε followed by incubation with HRO-labelled goat anti-mouεe IgM conjugate in the presence (solid line) or absence (broken line) of 4% (w/v) PEG 6000. Colour was developed using tetramethyl benzidine as substrate. Measurements were done in triplicate with standard deviation at <10%. Monoclonal culture supernatants were: AJ2 (•) , AJ3 (■) and AJ5 (*) . Figure 2 showε the reεultε of competitive ELISA (CELIA) uεing MAB AJ2. The ability of a variety of antigenε to inhibit MAB AJ2 binding to immobiliεed fetal εAChE was tested. AChE activity, aε determined by the Ellman aεsay, of the antigens were as follows: CSF-120 m units/ml; fetal brain DS-AChE-820 m units/ml; PεChE-224 m unitε/ml; and NHS-53 unitε/ml. AJ2 was used at a dilution of 1:250. Assays were done in triplicate and the εtandard deviation was not greater than 8%. Microtitre wells coated with FsAChE (200 ng/ml, 100ml) were incubated with MAB AJ2 culture supernatant (1:250 dilution) in the presence or absence of dilutionε of competing samples. Thiε was followed with HRO-labelled goat anti-mouεe Ig conjugate, then, the εubεtrate TMB. Reεultε were the average of triplicate meaεurementε and are expressed aε O.D. in the preεence of sample x 100 O.D. in the preεence of buffer Samples were (*) RBC-AChE, DS-AChE, PsChE and NHS. (■) AsAChE (•) FsAChE (A) CSF x-axiε AChE concentration scale refers to FsAChE, AsAChE. RBC AChE and PsChE, while the dilution scale refers to CSF, NHS and Ds-AChE. Figure 3 showε the reεultε of CELIA uεing MAB AJ5 at a dilution of 1:25. Experimental detailε are aε for Figure 2. Figure 4 εhowε the reεultε of CELIA uεing MAB AJ3 at a dilution of 1:50. Experimental detailε are aε for Figure 2. Figure 5 shows the results of an antibody capture sandwich ELISA for sAChE which uses MAB AJ2. Comparison of the binding of different antigens in the sandwich ELISA is εhown. Activities of AChE (m units/ml) in the antigen preparationε were: fetal brain homogenate-42.6; poεitive • amniotic fluid-62.2; and CSF-120. Microwell titre plateε were coated with goat anti-mouεe μ chain antibodieε (5μg/ml, 100 μl) and then incubated with MAB AJ2 culture supernantant for 5h at room temperature. After washing, dilutions of εampleε were added and incubation continued for 18h at room temperature. Next, rabbit anti-AChE antiεeru waε added (2h.room temperature) followed by HRO- labelled goat anti-rabbit IgG conjugate, and substrate TMB. O.D. was read at 450 nm. Samples were, fetal brain homogenate (■) , (+) amniotic fluid (•) , and cerebrospinal fluid (_). All assayε were done in triplicate with S.D. <10%. Figure 6 εhowε the reεultε of an antibody capture εandwich ELISA for sAChE uεing MAB AJ2. Comparison of binding of maternal serum from pregnancies from a non-NTD fetus (-) and pregnancieε from a NTD fetus (+) is shown. Asεayε were done in triplicate and the standard deviation waε <10-%. Experimental details are as for Fig 5, but uεing the following sampleε: (+) maternal sera A (■) , B (•) . (-) maternal sera F (D) , G (o) . REFERENCE EXAMPLE: Isolation and Partial Characteriεation of Human Adult and Fetal sAChE 1. EXPERIMENTAL PROCEDURES Materialε All che icalε were of analytical grade and purchaεed from Sigma Co., Poole, U.K. Biological material Cerebroεpinal fluid (CSF) and human adult brain were obtained from the Department of Clinical Chemiεtry, South ead Hoεpital, Briεtol, U.K. Human fetal brainε were diεεected from 12-18 week old aborted fetuεeε. In both caεeε tiεεueε were frozen at -70 ° C within Ih of diεεection. Preparation of tiεsue homogenate Extraction of εoluble AChE waε carried out by thawing the brain tiεεue and homogenising in 10 volumes (adult brain) or 5 volumes (fetal brain) per weight of ice-cold 0.3M sucrose containing 2 mM EDTA, in a Waring blender (5 x 30s) at 4*C. The homogenate was subjected to two cycles of freezing and thawing to disrupt the membranes and then centrifuged at 100,000 x g for 90 min at 4 β C. The supernatant was removed, aliquoted and stored at -70°C until required. Gel filtration of tissue supernatant Gel filtration waε carried out on Sephacryl (Trade Mark) S-200 (Pharmacia, Uppsala, Sweden) at 4*C. Supernatant εampleε (40 ml) were applied to a 2.5 cm x 100 cm column equilibrated in phoεphate buffered saline, pH 7.3 (phosphate buffered saline (PBS), 142 mM NaCl, 2.7 mM KC1, 8 mM Na 2 HP0 , 1.47 mM KH 2 P0 4 ) containing 0.02% NaN 3 . Elution was by upward flow at a rate of 50 ml/h. Fractions (5 ml) were collected and monitored for (i) protein content by measuring the optical density at 280 nm and (ii) acetylcholinesterase activity by the Ellman asεay (εee below) . The column waε calibrated with a Pharmacia High Molecular Weight kit. Affinity purification of sAChE sAChE in the gel filtration eluant was purified further by affinity chromatography on a column of edrophonium chloride-epoxy-Sepharose gel (Pharmacia) prepared according to Hodgson and Chubb (J.Neurochem..41,654-662.1983) . Samples (25 ml) were recycled through a 1 cm x 4 cm column equilibrated in PBS, at a flow rate of 15 ml/h for 24h at 4°C. Unbound material waε eluted with PBS containing 0.5 M NaCl (40 ml). The bound material waε then eluted with the εame buffer containing 12 mM edrophonium chloride (25 ml) . Edrophonium chloride waε removed by gel filtration on a Sephadex (Trade Mark) -G50 fine (Pharmacia) column (2.5 cm x 50 cm) equilibrated in ammonium acetate (50 mM) . Fractionε containing AChE activity aε determined by the Ellman aεsay were pooled, lyophiliεed, reconεtituted in PBS (2 ml) and aliquotε frozen at -20*C until required. Enzyme assays Cholinesteraεe activity waε meaεured at 30'C by the method of Ellman et ai. (Biochem.Pharm. ,2,88-95,1961) . Enzyme activity iε expreεεed aε IU (micromoles product formed per minute) . Acetylthiocholine iodide (1 mM) was uεed aε εubstrate. AChE activity was taken as that activity which waε inhibited by l,5-bis-(4-allyldimethyl- ammonium phenyl)-pentane-3-one dibromide (BW 284 C51; 1.5 μM) , while pεeudocholineεteraεe (PεChE) activity waε eεtimated aε the difference between two reaction mixtureε, one containing eεeriηe (20 μM) , the other BW 284 C51 (Hodgεon and Chubb, 1983) . Gel electrophoreεiε analvεiε of AChE activity AChE activity in different εampleε waε located on 6% polyacrylamide slab gels (8 cm x 8 cm) by the histochemical method of Coupland and Holmes (Q.J.Microsc.Sci. ,£8.,327-330, 1957) , using acetylthiocholine iodide (4 mM) aε substrate. The specificity of AChE activity waε establiεhed by inhibition with BW 284 C51 (1.5 x 10~ 4 M) . SDS-polvacrylamide σel electrophoresis (SDS-PAGE) SDS-PAGE was performed esεentially according to Laemmli (Nature.227.680-685.1970) with 8% polyacrylamide εlab gelε (16 cm x 18 cm) . Protein waε stained with silver (Nielsen and Brown,Anal.Biochem. ,112,311-315,1984) . The molecular weight reference standardε used were: £-galactosidaεe (120,000), phoεphorylaεe b (97,000), human IgM μ chain (74,000), bovine ε . erum albumin (65,000), human IgG chain (50,000), aldolaεe (40,000), carbonic anhydraεe (29,000) and human light chain (23,000). Sucroεe denεity gradient centrifugation The εedimentation coefficient of sAChE (300 μl) waε estimated in 4-20% (w/v) sucroεe gradientε (5 ml) in 50 mM εodium phosphate buffer, pH 8.0, using β- galactosidase (15.9 S) , catalase (11.3 S) and glucoεe oxidaεe (7.9 S) , aε sedimentation markers. Centrifugation was performed at 4°C in a Beckman L5-50 ultracentrifuge with a SW50 rotor (15h at 150,000 xg) . Fractionε (250 μl) were recovered from the gradient and assayed for AChE activity by the Ellman assay. Isoelectric focusing (IEF) Analytical isoelectric focusing waε carried out at 10*C on 5% polyacrylamide - 0.5% agarose cylindrical gels (0.4 cm x 8 cm) aε deεcribed by Wrigley (Meth.Enzvmol..22. 559-564,1971). Ampholyteε used were LKB, pH range 3 - 10 and 4 - 6. Following electrofocusing, gels with sAChE εa ples were histochemically stained for enzyme activity as described above. The pi of the enzyme waε determined from a εtandard plot conεtructed by electrofocusing a number of marker proteins of known pi (IEF calibration kit, Sigma Co. ) Lectin Binding The interaction of sAChE with lectins waε inveεtigated by affinity chromatography on Agaroεe-bound lectinε. The -following reεinε were uεed (Sigma Co. ) :Agarose-Concanavalin A type III-A, 10 g lectin/ l gel; Agarose-Lenε culinariε 2.5 mg lectin/ml gel and Agaroεe-ricinuε communiε, 2.5 mg lectin/ml gel. The gel (100 μl) waε washed by centrifugation (three times, 1 ml each) with 50 mM sodium acetate buffer, pH 7.0, containing 0.3M NaCl and 2.5 mM of each of the following salts, CaCl 2 , MgCl 2 , MnCl 2 and ZnCl 2 . sAChE (10 m unit) waε added in the same buffer (400 μl) and the suεpenεion waε incubated for 4h at 4 C C with gentle mixing. The gel waε waεhed by centrifugation (three timeε, 0.5 ml each), and the enzyme activity in the unbound material was meaεured by the Ellman aεεay. Non-εpecific binding waε determined by uεing Agaroεe-bound goat immuno- globulin G gel (Sigma Co.). Protein Estimation Protein concentration was determined by uεing a modified Lowry aεsay (Markwell e_t a_l. , Anal.Biochem..87.206-210. 1978) . Bovine serum albumin was used as a standard. 2. RESULTS Extraction of soluble AChE 20 - 30% of the total AChE activity in adult and fetal brains waε recovered in the high speed supernatant. Enzyme activities and protein contents are shown in Table 1. t gh-speed AChE and ChE activities were measured by the Ellman asεay uεing acetylthiocholine iodine (1 mm) aε a substrate and BW 284 C51 (1.5 μm) aε AChE inhibitor. Protein concentration waε estimated by Lowry. Protein concentrations and enzyme activity varied by <20% between different preparations. Analyεiε of the enzyme activity by PAGE εhowed that both εupernatantε contained three bandε of eεteraεe activity: a εlow moving PεChE band which waε not inhibited by BW 284 C51, an intermediate and a faεt moving AChE band, both of which were inhibited by BW 284 C51. Gel Filtration Brain εupernatant waε fractionated by gel filtration on a Sephacryl S-200 column. Three major protein peakε were obtained, eluting at molecular weights, >250,000 (void volume), 50,000 - 60,000 and <10,000, respectively. AChE activity in both supernatants was fractionated into two well-resolved peaks, PI and P2, with elution volumes corresponding to approximate molecular weights of 220,000- 240,000 and 45,000-55,000 daltons, respectively. Fractionε correεponding to each peak were pooled. In the adult brain supernatant, the applied AChE activity was equally divided into the two peakε, while >70% of the fetal supernatant AChE activity appeared in peak PI. Analysis of esteraεe activity by PAGE showed that PI contained most of the slow moving AChE (which correspondε to sAChE in CSF) as well aε PεChE and some of the fast moving AChE. P2 contained some sAChE, a negligible amount of PεChE and oεt of the faεt moving AChE. Affinity chromatography of AChE Adult and fetal PI and P2 were subjected to affinity chromatography on edrophonium-Sepharoεe gelε. AChE activity in the applied sampleε, unbound fraction and the edrophonium chloride eluted fraction waε determined by the Ellman aεεay. Between 60-80% of the AChE activity in PI bound to the affinity column, 40-50% of which could be εubεequently eluted with edrophonium chloride, while leεε than 3% AChE activity in P2 bound. Analyεis of the edrophonium chloride-eluted fraction by PAGE showed the presence of one band of enzyme activity which waε completely inhibited by BW 284 C51 and had an electro- phoretic mobility correεponding to that of sAChE present in CSF. Protein staining of PAGE showed the presence of one band in the edrophonium chloride fraction corresponding to the poεition of the AChE band. The edrophonium chloride fraction will thereafter be termed sAChE. The specific activity of the purified enzyme following desalting on the Sephadex G50 column was 500-800 units/mg, and 900-1500 units/mg for adult and fetal sAChE, respectively. However, both preparations lost activity rapidly. Hence, following freeze-drying and reconεtitution in PBS, the specific activity decreased to 50-100 units/mg. The drop in activity continued even on storage at -20*C. A similar inactivation of the enzyme upon purification was reported for fetal bovine serum sAChE (Chubb et al., Neuroscience, 10(4) ,1369-1377,1983) , and brain AChE (Gordon _Bt a_l. , Biochem.J. ,152,69-76,1976) . SDS-PAGE Analyεiε of adult .and fetal sAChE by SDS-PAGE under reducing conditionε showed the presence of a predominant band at M r 66,000. There waε no difference in molecular weight between the adult and fetal enzymeε. Sucroεe-density gradient centrifugation Adult sAChE revealed two major forms sedimenting at 15.3 S 4W and 10.4 S 4W . The former is probably aggregated material. A minor peak with a sedimentation constant of 6.25 S 4W iε alεo apparent. Fetal sAChE alεo revealed three forms of AChE activity, though with different relative diεtribution, at 15.0 S 4W , 10.5 S 4W and 6.1 S 4W . The 6.1-6.2 S form in both caεeε increaεed on εtorage, even at -20 C C. The differenceε in sedimentation coefficients between the adult and fetal formε of AChE were not significant. Iεoelectric focuεing of εAChE Electrofocuεing of purified sAChE waε performed using ampholyteε with pH ranges of 3-10 and 4-6. Enzyme staining gels revealed one band of activity which was completely inhibited by BW 284 C51. The pi valueε obtained (n = 10, mean ± S.D.) were: adult sAChE 5.47 ± 0.22, and fetal sAChE 5.47 ± 0.17. Lectin binding Adult or fetal sAChE (10m units of each) were mixed with the lectin agarose for 4h at 4'C. After waεhing, the amount of unbound enzyme in the eluant waε determined by the Ellman assay. The results are shown in Table 2. Values are the average of five experiments with the range in brackets. Table 2 Binding of εAChE to Lectin columns % sAChE bound Lectin specificity adult εAChE fetal sAChE Con-A mannose and glucoεe 92 (85-98) 95 (84-97) Lentil mannoεe and glucoεe 97 (83-99) 98 (95-99) Ricinuε galactoεe 82 (75-89) 88 (80-90) IgG nil 6 ( 3-8 ) 5 ( 3-9 ) Table 2 εhowε that over 80% of both adult and fetal εAChE waε absorbed onto the lectin resins used. Thiε interaction was specific aε shown by the lack of binding to Agaroεe - IgG resin. The binding of εAChE to the lectinε was very εtrong and no more than 50% of the bound enzyme could be deεorbed with the correεponding ligand (0.5M α-methyl glucoεide, 0.5M α-methyl mannoεide and 0.25M 3-methyl galactoεide) . EXAMPLE 1 !• Methods: Purification of human fetal and adult εAChE: deεcribed in the Reference Example. Production of polyclonal antiεera: Rabbitε were immuniεed by intramuεcular injection with adult sAChE (50 μg) in Freund's Complete Adjuvant (FCA) . After 4-5 weekε, the rabbitε were injected similarly and bled 5 dayε later. Antiserum was aliquoted and stored at -20°C. Production of monoclonal antisera: Three fuεionε were performed uεing fetal sAChE: (a) I_n Vivo immunisation: A Balb/c mouse waε immuniεed intraperitoneally with fetal sAChE (50 μg) in FCA. After 4 weeks, the mouse was injected intravenously with 25 μg of the antigen and fusion with the myeloma cellε (X63/Ag8.653) was performed four days later essentially aε described by Fazekes and Scheidegger (J.Immunol. eth..35.1.1980) . (b) I_n Vitro immunisation: Spleen cells from an unprimed Balb/c mouse were incubated at a cell density of 1,000,000 cells/ml of medium containing fetal sAChE (50 ng) per ml. The culture medium used was a 50:50 mixture of normal culture medium (10% FCS in RPMI1640 supplemented with glutamine (2 mM) and 2-mercaptoethanol (50 μM) ) , and thymocyte-conditioned medium (see below) . After 5 dayε in culture the εplenocyteε were harveεted and fused with X63 myeloma cellε aε deεcribed before. Thymocyte-conditioned medium waε prepared aε followε; Thy uε glands were diεsected out of five 12-14 dayε old Balb/c mice. A εingle cell suspenεion waε made and the cellε were incubated for 3 dayε at 37 β C at a cell denεity of 5,000,000 cellε/ml in 20% FCS in RPMI1640 medium containing glutamine (2 mM) and 2-mercaptoethanol (50 μM) . After 3 dayε the cellε were centrifuged and the supernatant (thymocyte-conditioned medium) waε filtered (0.22 μM) and εtored frozen at -70 β C. (c) I_n Vivo + In Vitro immunisation: A Balb/c mouse waε immuniεed with fetal sAChE as in (a) . After 2 weekε, the εpleen waε removed and cellε subjected to In Vitro immunisation aε described in (b) . Screening of hybrido as: Culture supernatants were initially screened using an ELISA. Microtitre wells were coated with fetal sAChE (200 ng/ml) in 50 mM sodium carbonate/bicarbonate buffer, pH 9.5 overnight at 4 * C. After washing with assay buffer (50 mM Tris-HCl, 0.15 M NaCl, 1.0% casein, 0.05% Tween-20, pH 7.2), 100 μl of culture supernatant was added to each well followed with 100 μl of aεεay buffer. The plate was incubated overnight at 4 β C. Following washes (3x) with asεay buffer, 100 μl of horse-radish peroxidase labelled goat anti-mouse immunoglobulin antiserum appropriately diluted in asεay buffer were added per well. After lh incubation at room temperature, the plate waε washed and subεtrate added (tetramethylbenzidine) . Colour intenεity waε meaεured after 30 min at O.D.450. Modified ELISA: the aεεay waε performed aε above but with the addition of polyethyleneglygol (PEG6000) at 4% (w/vol.) to the εecond antibody step. Determination of monoclonal antibody (MAB) specificity: MAB to ' sAChE were characteriεed by uεing the following methodε: (a) Nitrocelluloεe Immunodot Binding: Different putative antigen solutions (2 μl) were spotted onto nitro¬ cellulose paper. After drying and blocking with asεay buffer, culture εupernatantε in aεεay buffer were added and incubation with shaking waε performed at room temperature for 4h. The paper was washed with aεεay buffer and then incubated with goat anti-mouεe immunoglobulin-horεe radiεh peroxidaεe (HRO) conjugate for 2h at room temperature. The paper waε washed as before and incubated with substrate solution (3-amino-9-ethyl carbazole) for 30 min. (b) Competitive ELISA (CELIA) : This waε performed aε the modified ELISA but with one change. The culture εupernatant at the appropriate dilution were pre-incubated overnight at 4 • C with the unknown prior to addition to the microtitre wellε. (c) Immunoaffinity Chromatography: The MAB waε covalently bound to solid phase as followε: Culture εupernatant (50 ml) waε mixed with Sepharoεe-4B-goat anti- mouεe IgM antibody gel (1 ml) (Sigma Co.) for 2h at room temperature followed by overnight incubation at 4*C. Unbound material waε washed away and " the IgM-anti-IgM complex was cross-linked as followε: the gel-antibody complex waε incubated with 0.2 mg/ml dimethyl suberimidate in 0.2M triethanolamine buffer, pH 8.4 for 3h at room temperature to covalently cross-link the MAB to the anti- IgM on the Sepharoεe gel. The reaction waε terminated by the addition of 0.5M lysine. The gel was washed with PBS and stored in the same buffer containing 0.2% sodium azide. Immunoaffinity absorption of AChE: Sepharose-MAB gel (0.2 ml) waε mixed with variouε antigen εolutionε made up to 500 μl in PBS. Incubation waε carried out for 2h at room temperature, then overnight at 4*C. Triton X-100 (0.1% (w/v)) waε added to the detergent-εoluble antigenε. After incubation, the gel and any bound antigen were spun down at low speed and the gel waε waεhed twice with PBS. All unbound material waε kept for analysis by the Ellman enzyme asεay and by PAGE. Enzyme assay for AChE: ChE and AChE activity was assayed by the Ellman test. PAGE analvsiε of ChE and AChE: Polyacrylamide gel electrophoreεis waε performed aε deεcribed by Coupland and Holmeε (Q.J. Microεc.Sci. ,98, 327-330) . Sandwich ELISA for AChE: Microtitre wells were coated overnight with goat anti- mouse IgM antibodies (Sigma) (5 μg/ml, 100 μl) overnight at 4 * C. After washing and blocking with assay buffer, 100 μl of dilutions of culture supernatant was added and the plate incubated for 5h at room temperature. After washing, samples containing antigens were added and incubation carried out overnight at room temperature. The plate was washed and an appropriate dilution of rabbit anti-AChE antiεerum in aεεay buffer waε added. After 2h incubation, HRO-labelled goat anti-rabbit IgG waε added followed by the substrate. In an alternative asεay, following the incubation with the antigen solution, biotin-labelled rabbit anti-AChE immunoglobulin fraction was added at the appropriate dilution for 2h, followed by horse-radish peroxidase labelled εtreptavidin (Sigma) , then by the substrate. 2. RESULTS Purification of AChE: described in the Reference Example. In summary, purified fetal and adult sAChE were obtained from human brain. Gel filtration, sucrose-denεity gradient centrifugation and SDS-PAGE analyεeε indicate that the enzyme iε a tetramer with a molecular weight of around 240,000 (10.15 S) conεiεting of four εubunitε of molecular weight of 66,000. The enzyme haε a pi of 5.47. Polyclonal anti-εAChE antisera: Four rabbit antisera were raised to the adult form of εAChE. Analysiε by CELIA εhowed them all to croεε react with other AChE formε. Theεe antiεera were uεed later on in εetting up εandwich ELISAε. Monoclonal anti-εAChE antibodieε: Both fuεion experiments (a) and (b) (see Methods section) produced more than 90% of wells positive for hybrido as, but no anti-AChE antibodieε were detected. Hence, it was decided to use a combination of I_n Vitro and In Vivo immunisations (fusion experiment (c) ) . The modified ELISA waε used aε a screening aεsay. Addition of PEG to the MAB incubation step, i.e. to the step of incubating a culture supernatant with fetal sAChE-coated microtitre wells, actually reduced the sensitivity of the ELISA to detecting MABs specific for fetal sAChE. However, PEG addition to the second antibody incubation step at 4% (w/v) greatly enhanced the detection limit. Figure 1 shows the remarkable effect of PEG on the titration of three MAB to fetal sAChE. This experiment waε done uεing MAB from hybridomaε that had been cloned three timeε and whoεe antibody content waε quite high. Addition of PEG waε routinely done to all subsequent asεayε. Fuεion experiment (c) produced a number of poεitive hybrids. Nine of theεe were cloned three timeε by limiting dilution. Reεults obtained with εome of theεe are reported here. All the nine MAB were of the IgM claεs. A test of specificity for six of theεe MAB waε effected by the dot blot method. All εix MABε εhowed a preference of binding to the ' fetal sAChE to which they were raiεed. Characteriεation of MABε: bv CELIA: ELISA titration curveε of culture εupernatantε were obtained for the nine MABε. The εpecificity of theεe were checked by the more sensitive CELIA. Figures 2, 3 and 4 illustrateε the reεultε obtained with MABs AJ2, AJ5 and AJ3, respectively. All three MABs did not crosε react with human RBC-AChE, pChE, normal human serum (NHS) and detergent-soluble membrane AChE from human fetal brain. All these are known not to contain sAChE. The strongest reactivity waε obtained with purified fetal sAChE and less reactivity with purified adult sAChE, and CSF (which contains only adult sAChE) . Characterisation of MAB bv Immunoaffinitv Chromatography: The specificity of MAB to sAChE was further tested by immunoaffinity absorption. The MABs were immobilised and covalently-linked to Sepharose-4B-anti-IgM gel for two reasonε: (i) to allow multiple use of the gel and (ii) the covalent cross-linkage waε found to stabiliεe antibody activity of the MAB. Resultε of all the binding studies carried out with MAB AJ2, AJ3 AND AJ5 are shown in Table 3. The Table shows the percentage of enzyme activity in the samples which bound to each MAB affinity column. The amount of activity bound to the edrophonium-Sepharose column iε shown for comparison. Table 4 shows the AChE and pChE enzyme activities of the antigen εolutionε uεed' in the binding studieε. Theεe activitieε were determined by the Ellman aεεay. TABLE 3: Immunoaffinity Absorption Chromatography Antigen AJ2 Ab column AJ3 mAb column AJ5 mΛb column Edrophonium- Sepharose colum Fetal brain 67% AChE 65% AChE 69% AChE 61% AChE homogenate 0 PsChE 0 PsChE 0 PsChE 0 PsChE Human serum PsChE NHS 0 0 AChE 7% PsChE Detergent- 0 9% AChE soluble AChE 0 PsChE Red blood cell AChE CSF 48% AChE not tried not tried 85% AChE 0 PsChE 0 PsChE Table : Activities of the variouε antigen solutions Antigen AChE activity PsChE activity (m units/ml) (m units/ml) Adult brain homogenate 43.0 11.0 Fetal brain homogenate 42.6 34.0 CSF 120.0 50.0 Detergent- soluble AChE (fetal) 820.0 48.2 Human serum PsChE 224.0 1970.0 Human red blood cell AChE 1000.0 120.0 NHS 53.0 658.6 Amniotic fluid 62.2 87.5 It can be εeen from Table 3 that only εAChE iε bound to MAB AJ2 and MAB AJ3 columnε. RBC AChE, pChE, detergent εoluble brain AChE and non-secretory type soluble AChE did not bind to these columnε. In fetal brain homogenate, 67% of the AChE activity waε bound by the AJ2 column, which compareε well with the 61% AChE bound from thiε material by the edrophonium-Sepharose column. All thiε bound AChE iε preεumed to be sAChE, as the edrophonium-Sepharose gel iε specific for sAChE. Binding of sAChE in CSF to the AJ2 column waε alεo carried out by the εame method and 48% bound aε oppoεed to 85% binding to the Edrophoniu - Sepharoεe gel. Thiε again εuggeεtε that Mab AJ2 poεεibly has greater affinity for εAChE from a fetal, rather than adult, origin. Im unoassav for sAChE: A sandwich ELISA waε εet up uεing MAB AJ2 and the polyclonal rabbit anti-AεAChE antiεerum. Thiε aεεay waε then used with some AF samples and maternal sera obtained from pregnancies with known outcome. Figure 5 showε titration curveε for fetal brain homogenate, CSF and a poεitive (+) AF (AF from an NTD pregnancy) . It can be clearly seen that the asεay can detect εAChE in (+) AF. The Figure alεo indicateε that the greateεt activity iε found in the CSF and the leaεt in the fetal brain homogenate. This iε different from the results obtained with CELIAs, where reεultε indicate greater activity in the fetal brain homogenate than in the CSF. The reaεon for this, is most likely to do with aεεay format. In the Sandwich ELISA the detection relieε on the uεe of rabbit anti-AChE antiserum. This, aε explained earlier, was raiεed to the adult sAChE. Therefore the results may reflect greater affinity of this antiserum to the adult rather than the fetal form of sAChE. This assay using HRO-labelled anti-rabbit IgG aε the revealing syεtem waε uεed with a number of (+) AF and (-) AF εampleε. Table 5 εhowε the reεultε obtained. Meaεurementε were done in triplicate and the εtandard deviation waε <10%. Both (+) and (-) samples showed high absorbance. However, the average absorbance given by the (+) AF samples was markedly higher than that of the (-) AF εampleε. The εame aεεay waε applied to a (+) and (-) maternal sera. Figure 6 shows the titrations obtained with two εampleε of each. Once again both gave high abεorbance readingε. Improvement of the aεεay was attempted uεing a biotin/ εtreptavidin revealing εyεtem. Thiε gave better reεultε in term of low background and εenεitivity. Table 6 εhowε the reεults obtained with (+) and (-) AF and maternal εera εampleε. ______________: Antibody capture εandwich ELISA uεing MAB AJ2 Amniotic Fluid Optical denεity 450nm Sample Dilution of εample 1.15 0.91 0.68 0.52 1.06 0.91 0.53 0.19 Table 6 Sample Optical density Average O.D. 450nm 0.76 0.61 C 1.57 1.30 1.21 1.36 A 1.94 1.79 1.53 1.75 1.42 D 1.20 1.11 1.10 1.14 F 1.80 1.45 1.51 1.42 G s (-) 0.67 1.02 0.85 0.85 F s (-) 0.58 0.80 0.91 0.76 0.76 H ε (-) 0.71 0.81 0.67 0.73 J s (-) 0.71 0.76 0.61 0.69 Blank value (n = 3) 0.64 AF+ amniotic fluid positive for sAChE AF- amniotic fluid negative for sAChE S+ serum from a known NTD pregnancy S- serum from a known non-NTD pregnancy EXAMPLE 2 Materialε All chemicalε were of analytical grade and purchaεed from Sigma Co, Poole, U.K. Biological materialε were obtained aε deεcribed in the Reference Example. Preparation of tiεεue homogenate Extraction of fetal εoluble AChE was carried out by thawing the brain tiεεue and homogeniεing in 5 volumeε (fetal brain) per weight of phosphate buffered saline (PBS) containing 2 mM EDTA, in a Waring blender (5 x 30s) at room temperature. The homogenate was subjected to two cycles of freezing and thawing to disrupt the membranes, diluted 4- fold with PBS containing 2mM EDTA and then centrifuged at 18,000 x g for 90 min at 4°C. The εupernatant waε removed, aliquoted and εtored at -70°C until required. Affinity purification of εAChE sAChE waε purified from the fetal brain homogenate by direct affinity chromatography on a column of edrophonium chloride-epoxy-Sepharoεe gel (Pharmacia) prepared according to Hodgson and Chubb (J. Neurochem., 4_1, 654-662, 1983). Up to 500 ml of fetal brain homogenate were recycled through a 1 cm x 7 cm column equilibrated in PBS, at a flow rate of 1 ml/min for 24 h at room temperature. Unbound material waε eluted with PBS containing 0.5 M NaCl (35 ml). The bound material waε then eluted with the same buffer containing 12 mM edrophonium chloride (35 ml) . Edrophonium chloride waε removed by gel filtration on a Sephadex (Trade Mark) -G50 fine (Pharmacia) column (2.5 cm x 50 cm) equilibrated in ammonium acetate (50 mM) . Fractionε containing AChE activity aε determined by the Ellman assay were pooled, lyophilised, reconstituted in PBS (2 ml) and aliquots frozen at -20°C until required. Thiε preparation gave pure fsAChE as determined by SDS- PAGE, visualised by silver staining. Fetal calf serum AChE (FCε AChE) waε prepared in the εame way, except the fetal calf serum (Gibco, U.K.) waε recycled through the column up to 100 ml at a time. Production of FCS AChE. or fetal secretary. AChE (human) carrier protein conjugates 20 μg of AChE waε coupled to 200 μg of keyhole limpet hemocyanin (KLH) (Pierce Co, England) by incubation with 0.5 ml of 0.1% glutaraldehyde in PBS at room temperature for 16 hr. The produced waε dialyεed verεuε PBS to remove any unreacted glutaraldehyde. Production of monoclonal antibody hybridomaε) Eight fuεionε were performed uεing fetal εAChE: (A) Fusion l: Immunisation with fetal εAChE coupled to KLH. An Fl hybrid mouεe (CFLP x Balb/c crosε) waε immuniεed by εubcutaneouε inoculation with fetal εAChE/KLH conjugate (60μg) in Freundo Complete Adjuvant. After 8 dayε the mouεe waε booεted by inoculation into the peritoneal cavity of 60μg of fetal εAChE/KLH conjugate in Freundε Incomplete Adjuvant. After 6 dayε 5 μl of blood was taken by εuperficial veneεection tail and aεεayed by direct ELISA for reactivity of antibodieε with fetal sAChE aε coating antigen. The antisera proved positive even when diluted down by l/io 5 . Four dayε later a further inoculation of 60μg of fetal sAChE/KLH conjugate was made into the peritoneal cavity in PBS buffer. 7 dayε later a final booεter inoculation into the peritoneal cavity waε made of 30 μg fetal εAChE/KLH conjugate in PBS. After 4 dayε splenocytes were removed and fused with myeloma cells (x63/Ag8.653) essentially as described by Fazekas and Scheidegger (J. Immunol. Meth. , 35, 1, 1980). (B) Fuεionε 2-4 and 7-8 Intraεplenic immunisation: Three Fl hybrid mice (CFLP x Balb/c crosε) - Fuεionε 2-4 and two Balb/c mice - Fusions 7 and 8 were anaesthetised with hypnorm/midazolam. The fur waε clipped over the spleen and an incision 1-1.15 cm long waε made juεt below the left set of ribs. The muscle was separated by blunt dissection, and the lower pole of the spleen was exteriorised through a small incision made in the peritoneum. Fetal sAChE 9μg in 50μl PBS waε inoculated directly into the spleen via a 27g x 27cm (1/2 inch) needle. The spleen waε puεhed back into the peritoneal cavity, and inciεion waε closed using metal stitch clips. Splenocytes were removed after 4 dayε and fuεed with myeloma cellε (x63/Ag8.653) eεsentially as described by Fazekas and Scheidegger (J. Immunol. Meth, 35, 1, 90). (c) Fusions 5 and 6: In vitro immuniεation . (i) Thymus conditioned medium waε prepared by forming a mixed thymocyte culture of thymocyte from 6 three week old Balb/c mice and from 6 three week old CFLP mice. They were cultured for 24 hrε or 48 hrε in Dulbeccoε Modified Eagleε Medium (DMEM) with 5% (v/v) Myoclone fetal calf serum + 5 x 10~ 5 M 2-mercaptoethanol + HT supplement (Sigma) + 18mM HEPES pH 7.3. Cells were cultured at a denεity of 5 x 10 6 /ml in 25 cm 2 flaεkε. The supernatant waε harveεted, filtered (0.22μm) and stored at -20°C. (ii) EL4 supplement. EL4 cells (obtained from ECACC, Porton Down) were cultured at 1 x 10 6 cells/ml in DMEM at 5% myoclone FC5 + 5 x 10~ 5 M 2-mercaptoethanol + HT supplement + 18mM HEPES pH 7.3 + long phorbol myristate acetate/ml for 40 hrs. The supernatant was harvested, filtered (0.22 μm) and stored at -20°C. In vitro culture Splenocytes were removed from an Fl mouse (Balb/c x CFLP cross) and cultured at 5 x 10 6 cells/ml in medium containing 0.8 μg/ml fetal sAChE. The medium used waε Dulbeccoε Modified Eagles Medium + 5% v/v myoclone fetal calf serum + 33% (v/v) thymus conditioned medium + 25% (v/v) EL4 εupoplement + 5 x 10~ 5 M 2-mercaptoethanol + HT εupplement + 18 mM HEPES pH 7.3. Two 25cm 2 flasks were set up. After 6 days stimulated cells from one flask were f εed with myeloma cellε (x63/Agδ.653) eεsentially as described by Fazekas and Scheidegger (J. Immunol. Meth. 35, 1, 90) . Thiε was fuεion 6. The cellε in the εecond flaεk were reεtimulated for a further 6 dayε with 0.8μg/ml fetal sAChE in fresh medium prior to fuεion with myeloma cellε by the εame method. Thiε waε fuεion 5. Screening of Hvbridoroaε Culture εupernatantε were initially εcreened uεing a direct ELISA. However due to the very low εignalε leading to spurious reεultε, cellε were either pulεed with serum- free medium at least 1 day prior to screening with the original direct ELISA or were grown solely in serum-free medium. This had the effect of making sure none, or very little (<1%) fetal calf serum waε preεent in the εupernatant at the time of screening. Direct ELISA for screening hvbridomaε Microtitre wells were coated with human fetal sAChE (200 ng/ml) in 50 mM sodium carbonate/bicarbonate buffer, pH 9.6 overnight at room temperature. Fetal sAChE when available in larger quantity waε used at 400 ng/ml. After washing thrice with asεay buffer (phosphate buffered saline, pH 7.2, 0.05% Tween-20) , 100 μl of culture supernatnat waε added to each well and the plate incubated for 2 h at 37*C. Following waεheε (5x) with assay buffer, lOOμl of horse- radiεh peroxidaεe labelled goat-anti mouεe immunoglobulin antiserum appropriately diluted in assay buffer was added per well. After 1 hr incubation at 37 β C, the plate waε washed (5x) with assay buffer and substrate added (tetramethylbenzidine solution) . After stopping the reaction with 50μl of 2M H 2 S0 4 , column intensity was measured at O.D. 450. Determination of monoclonal antibody (MAB) specificity bv ELISA Microtitre wells were coated with human fetal sAChE (400 ng/ml) or Red blood cell AchE (5μg/ml) in 50mM sodium carbonate/bicarbonate buffer, pH 9.6, overnight at 4'C 200μl/well at all stageε. After blocking for 30 min, and waεhing (2x) with aεεay buffer (50mM Triε/HCl, 0.05% Tween- 20, 0.02% sodium azide, pH 7.4), 200μl of each MAB diluted in aεsay buffer plus 0.1% casein waε added to each well and the plate incubated for three hourε at room temperature. Following waεhing (3 x 10 minε) with aεεay buffer, wellε were incubated with an alkaline phoεphate conjugated goat anti mouεe immunoglobulin conjugate appropriately diluted in aεεay buffer plus 0.1% casein plus 4% PEG 6000 for one hour at room temperature. Following waεhing with aεsay buffer substrate solution (lmg/ml p-nitrophenyl phosphate disodium in 10% w/v diethanolamine-HCl, 0.01% magnesium chloride, 0.02% sodium azide, pH 9.8) was added and incubated for 1 hr at room temperature prior to reading at 405 nm. RESULTS Hvbridomas secreting monoclonal antibodies reactive with fetal sAChE after cloning Asεayε were performed uεing the new direct ELISA from serum-free medium supernatant . Fuεion 1: Immunized and booεted in vivo with fetal sAChE coupled to KLH. From an Fl mouεe. Two cloned hybridomaε were created. AB 1-1 AB 1-7 Fuεion 2: Intraεplenic: Fl mouse. Two cloned hybridomas were created. AB 2-2 AB 2-3. Fuεion 3: Intraεplenic: Fl mouεe. Three cloned hybridomaε were created. AB 3-2 AB 3-5 (deposited at the European Collection of Animal Cell Cultures, Porton Down, GB on 3 December 1990 under acceεεion number 90120322. AB 3-7. Fuεion 4: Intraεplanic: Fl mouεe. One cloned hybridoma waε created. AB 4-3 AB 4-7. Fuεion 5: in vitro immuniεation, reεtimulated: Fl mouεe. Seven cloned hybridomε were created. AB 5-1 AB 5-7 AB 5-8 AB 5-9 AB 5-12 AB 5-14 AB 5-15 Fuεion 6: in vitro immuniεation, Fl mouεe. Five cloned hybridomaε were created. AB 6-2 AB 6-3 AB 6-5 AB 6-7 AB 6-10 Fusion 7: Intrasplenic immunisation Balb/c mouεe. Six cloned hybridomaε were created: AB 7-1 AB 7-3 AB 7-6 AB 7-7 AB 7-9 AB 7-10. Fuεion 8: Intraεplenic immuniεation, Balb/c mouse. Five cloned hybridomaε were created: AB 8-1 AB 8-2 AB 8-4 AB 8-5 AB 8-6. The specificity of monoclonal antibodieε, aε determined by ELISA, is εhown in Table 7 below. Reεultε are given aε OD 405. TABLE 7 Clone antibody FsAChE as antigen Red Blood Cell AChE name concentration as antigen or dilution 1st reading 2nd reading 1st reading 2nd reading AB 4-7, AB 5-12, AB 3-5 were from ammonium εulphate cutε of crude aεciteε AB 6-3 waε a euglobulin precipitation from crude aεciteε. Cells secreting antibody not tested yet or poεitive aεciteε not yet produced include AB 2-2, AB 3-2, AB 3-7, AB 4-3, AB 5-1, AB 5-7, AB 5-8, AB 5-14, AB 6^2, AB 6-5, AB 6-7, AB 7- 6, AB 8-4, AB 8-5, AB 5-6. Thiε experiment shows that specific antibody-secreting cellε have been produced from fuεion 3 (AB 3-5) fuεion 4 (AB 4-7), fuεion 5 (AB 5-12), fuεion 6 (AB 6-3), fuεion 7 (AB 7-3, AB 7-10) and fuεion 8 (AB 8-1).

Claims: CLAIMε 1. A monoclonal antibody which iε specific for human secretory acetylcholinesterase (sAChE) and which showε no cross-reactivity with other types of human acetyl- cholinesterase. 2. A monoclonal antibody according to claim 1, which is an IgM or IgG. 3. An immortalised cell line which secretes a monoclonal antibody aε claimed in claim 1 or claim 2. 4. A proceεε for the preparation of a monoclonal antibody as claimed in claim 1 or 2 , which proceεε compriεeε : (a) culturing an immortaliεed cell line which secretes the monoclonal antibody; and (b) isolating the monoclonal antibody thuε produced. 5. A proceέε for the preparation of an immortaliεed cell line aε claimed in claim 3, which proceεε comprises : (I) (a) immunising an animal with adult or fetal human εAChE; (b) obtaining lymphatic tiεsue cells from the immunised animal ; (c) culturing the obtained cells with adult or fetal human sAChe; (d) immortaliεing the cultured cellε; and (e) screening the resulting cell lineε for an immortalised cell line which secretes monoclonal antibody which iε specific for human sAChE; or (II) (a) obtaining lymphatic tissue cells from an aminal, (b) culturing the obtained cells with adult or fetal human sAChE, (c) immortalising the cultured cells, and (d) screening the resulting cell lineε for an immortalised cell line which secretes monoclonal antibody which iε specific for human sAChE; or (III) (a) obtaining lymphatic tissue cells from an animal. (b) culturing the obtained cellε with adult or fetal human sAChE, (c) culturing the cellε obtained in step (b) with fresh adult or fetal human εAChE, (d) immortalising the cells obtained in step (c) , and (e) screening the resulting cell lines for an immortalised cell line which secretes monoclonal antibody which is specific for human sAChE; or (IV) (a) immuniεing an animal with adult or fetal human εAChE by inoculation directly into a lymphatic organ or lymph node, (b) obtaining lymphatic tisεue cellε from the immuniεed animal, (c) immortaliεing the cellε obtained, and (d) screening the resulting cell lines for an immortaliεed cell line which εecretes monoclonal antibody which iε specific for human sAChE. 6. A procesε according to claim 5, wherein εplenocyteε are obtained in εtep (b) of procesε (I) or (IV) or in εtep (a) of proceεε (II) or (III) and the cultured cellε are fuεed with myeloma cellε in step (d) of procesε (I) or (III) or in εtep (c) of proceεε (II) or (IV) . 7. A proceεs according to claim 5 or 6, wherein resulting cell lines are screened by - contacting a solid support coated with adult or fetal human sAChE with supernatant from a culture of the cell line and with, in the presence of polyethylene glycol, a labelled antibody capable of binding to any monoclonal antibody in the supernatant which binds to the human sAChE; or - contacting a solid support coated with adult or fetal human sAChE with supernatant from a culture of each cell line grown in serum-free or low serum-supplemented medium and with a labelled antibody capable of binding to any monoclonal antibody in the supernatant which binds to the human sAChE. 8. A method of determining human sAChE in a sample, which method comprises carrying out a said determination using a monoclonal antibody as claimed in claim 1 or 2. 9. A method according to claim 8, comprising contacting a sample suspected of containing human sAChE with a said monoclonal antibody and determining whether the said monoclonal antibody has bound to any human sAChE. 10. A test kit suitable for use in determining fetal human εAChE, which kit compriεeε a monoclonal antibody aε claimed in claim 1 or 2 and means for determining whether the monoclonal antibody haε, in use, bound human sAChE. 11. A monoclonal antibody which iε specific for human - sAChE and which εhowε no crosε-reactivity with human red blood cell AChE.

Inventors: BRENNAND DAVID MARK, JEHANLI AHMED MOHAMMED TAKI, MCBRIDE BRIAN, BRENNAND, DAVID, MARK, JEHANLI, AHMED, MOHAMMED, TAKI, MCBRIDE, BRIAN
Applicants: AVALON BIOSCIENCES LTD, AVALON BIOSCIENCES LIMITED
Application year: 1990
Main classification: C12N5
IPC codes: C07K16, C12N5, C12P21