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WO1991008302A1 - Anticorps monoclonaux specifiques a l'acetyl cholinesterase - Google Patents

Anticorps monoclonaux specifiques a l'acetyl cholinesterase Download PDF

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WO1991008302A1
WO1991008302A1 PCT/GB1990/001886 GB9001886W WO9108302A1 WO 1991008302 A1 WO1991008302 A1 WO 1991008302A1 GB 9001886 W GB9001886 W GB 9001886W WO 9108302 A1 WO9108302 A1 WO 9108302A1
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sache
human
monoclonal antibody
fetal
ache
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Ahmed Mohammed Taki Jehanli
Brian Mcbride
David Mark Brennand
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AVALON BIOSCIENCES Ltd
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AVALON BIOSCIENCES Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes

Definitions

  • MONOCLONAL ANTIBODIES SPECIFIC FOR ACETYL CHOLINESTERASE This invention relates to monoclonal antibodies, to their preparation and to their use in immunoas ⁇ ay ⁇ .
  • NTDs Neural Tube Defects
  • CNS central nervous system
  • Encephalocele is relatively uncommon, with the closure defect occurring a little lower down the neuroaxi ⁇ . Babies do survive with little paralysis, and they usually grow up spastic, severely retarded and often blind.
  • 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.
  • NTD' ⁇ are diagnosed by: (1) Genetic counseling: 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.
  • Alpha-fetoprotein is a glycoprotein with a molecular weight of 70,000 Dalton ⁇ . It i ⁇ u ⁇ ually 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 fetuse ⁇ . Currently, its measurement by an immunoa ⁇ say i ⁇ the main diagnostic test, in conjunction with US, for NTD.
  • AFP i ⁇ mea ⁇ ured in maternal serum An elevated value is followed by US scanning, then by amniocente ⁇ i ⁇ .
  • the amniotic fluid (AF) i ⁇ then te ⁇ ted for AFP.
  • AF amniotic fluid
  • AFP levels in AF also vary with gestational age. Furthermore, a major problem with as ⁇ aying AFP in AF i ⁇ the likelihood of a fal ⁇ e 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.
  • 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 ponent ⁇ are expected to be found in the AF and to diffu ⁇ e into maternal serum. These could, therefore, be potential markers for NTD.
  • 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 choline ⁇ ter ⁇ into choline.
  • 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.
  • AChE i ⁇ membrane-bound and 100% of red blood cells AChE is membrane-bound.
  • the central nervous system soluble AChE i ⁇ in itself of multi- forms. The bulk of it is believed to be derived from the membrane-bound AChE as i ⁇ ⁇ hown by its behaviour in the presence of detergent ⁇ .
  • AChE neural soluble AChE
  • CSF mammalian cerebrospinal fluid
  • AChE inhibitor edrophoniu chloride edrophoniu chloride
  • the i ⁇ olation and partial characterisation of the secretory form of human brain AChE has been described (Gardner et a].., Biochem.Soc.Trans. , 14 ,1234- 1235,1986) .
  • AChE amniotic fluid
  • a monomer 4.0 S
  • a dimer 5.5 S
  • a tetramer (10.3 S)
  • the origin of the first two i ⁇ not clear.
  • the dimer i ⁇ very much ⁇ imilar to the dimer membrane-bound AChE found in red blood cell ⁇ (RBC) .
  • the tetramer form i ⁇ 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:
  • 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.
  • the main problem ⁇ 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.
  • 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 hybridoma ⁇ 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 ⁇ pecific for human ⁇ ACheE. The monoclonal antibody can be u ⁇ ed in immunoa ⁇ says for fetal and adult human ⁇ AChE, thu ⁇ enabling NTD ⁇ to be detected.
  • the present invention provides a monoclonal antibody which is specific for human sAChE and which shows no cros ⁇ -reactivity with other type ⁇ 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 comprise ⁇ :
  • the invention also provides an immortalised cell line which secretes *a monoclonal antibody ⁇ pecific for human ⁇ AChE.
  • a cell line can be prepared by one of four proce ⁇ e ⁇ :
  • Proces ⁇ (II) comprises: (a) obtaining lymphatic tis ⁇ ue cells from an animal,
  • step (c) culturing the cells obtained in step (b) with fre ⁇ h adult or fetal human sAChE,
  • step (d) immortalising the cells obtained in step (c) .
  • Process (IV) comprises:
  • ⁇ plenocyte ⁇ are obtained in step (b) of process (I) or (IV) or in step (a) of proces ⁇ (II) or (III) .
  • 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) .
  • resulting cell lines are screened by
  • the invention further provide ⁇ a method of determining human ⁇ AChE in a sample, which method comprise ⁇ carrying out a ⁇ aid determination u ⁇ ing a monoclonal antibody which i ⁇ ⁇ pecific for human ⁇ AChE and which shows no cros ⁇ - reactivity with other type ⁇ of human acetylcholine ⁇ tera ⁇ e.
  • the method may compri ⁇ e contacting a sample suspected of containing fetal human sAChE with a said monoclonal antibody and determining whether the said monoclonal antibody ha ⁇ bound to any fetal human sAChE.
  • the invention additional provide ⁇ a test kit suitable for use in determining fetal human ⁇ AChE, which kit compri ⁇ e ⁇ a monoclonal antibody which i ⁇ specific for fetal human sAChE and which shows no cros ⁇ -reactivity with other type ⁇ of human acetylcholine ⁇ tera ⁇ e and means for determining whether the monoclonal antibody ha ⁇ , in use, bound to human sAChE.
  • the monoclonal antibodie ⁇ are specific for human sAChE. They react specifically with adult or fetal human sAChE and show no cros ⁇ -reactivity with other types of human acetylcholinesterase. In particular, they show no reactivity with human red blood cell AChE, and preferably no cros ⁇ -reactivity with human serum AChE, human me brane- bound neuronal AChE, human muscle AChE and neuronal-soluble non-secretory AChE. The antibodies do not cro ⁇ -react either with human serum pseudo-ChE.
  • 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 i ⁇ obtained from human brain ⁇ .
  • Fetal human brain ⁇ are typically obtained from aborted fetu ⁇ e ⁇ over 16 week ⁇ old.
  • the brain ⁇ are typically stored frozen, thawed and homogenised in ice-cold 0.3M sucro ⁇ e containing 2mM ethylene dia ine tetraacetic acid (EDTA) or u ⁇ ing pho ⁇ phate buffered saline/2mM EDTA as the buffer.
  • EDTA ethylene dia ine tetraacetic acid
  • the homogenate may be subjected to one or more cycles of freezing and thawing, and then centrifuged.
  • the supernatant is removed and optionally i ⁇ ⁇ ubjected to gel filtration.
  • Sephacryl S-200 can be used for this purpose. Fractions are collected having a protein content a ⁇ determined by optical den ⁇ ity at 280 nm, and AChE activity a ⁇ determined by the Ellman a ⁇ say.
  • the ⁇ AChE in the ⁇ upernatant from centrifugation or in the fraction ⁇ collected during gel filtration i ⁇ then purified by affinity chro atography.
  • a column of edrophonium chloride-epoxy-Sepharo ⁇ e gel may be u ⁇ ed.
  • An immortalised cell line may be prepared by any one of proces ⁇ (I) to (IV) . Prior to immortalisation:
  • Proces ⁇ (I) A non-human mammalian ho ⁇ t 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 cell ⁇ or other lymphatic ti ⁇ sue or peripheral blood lymphocytes may be obtained from the immunised host. Proces ⁇ (II) .
  • Cell ⁇ of lymphoid origin such as splenocytes, lymph node cell ⁇ or other lymphatic tissue or peripheral blend lymphocytes may be obtained from a mammalian host such a ⁇ a ou ⁇ e or rat.
  • the cell ⁇ 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 ⁇ g/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.
  • the incubation last ⁇ for a minimum of 3 day ⁇ , for example 4-10 day ⁇ .
  • a suitable period may be 5 days or 6 days.
  • Proces ⁇ (III) Cells of lymphoid origin such a ⁇ ⁇ plenocyte ⁇ lymph node cell ⁇ or other lymphatic tissue or peripheral blood lymphocytes may be obtained from a mammalian ho ⁇ t such a ⁇ a mou ⁇ e or rat. The cell ⁇ are incubated in accordance with the procedure for proce ⁇
  • An in vitro boo ⁇ t may be given by tran ⁇ ferring the cell ⁇ to fre ⁇ h culture medium containing sAChE.
  • the culture medium may also contain thymocyte-conditioned medium, culture ⁇ upernatant from EL4 cells, phorbol myristate acetate or other growth factors.
  • 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.
  • a class switch from lgM to lgG antibody may be induced.
  • the culture cells are then immortalised.
  • Proces ⁇ (IV) A non-human mammalian host such a ⁇ a mou ⁇ e or rat i ⁇ 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.
  • a mouse i ⁇ anae ⁇ theti ⁇ ed with hypnorm/midazolan Typically a mouse i ⁇ anae ⁇ theti ⁇ ed with hypnorm/midazolan. The fur i ⁇ clipped over the spleen and an incision 1-1.5 cm long is made just below the left set of ribs. The mu ⁇ cle is separated using blunt dissection, and the lower pole of the spleen exteriorized through a small incision in the peritoneum.
  • lymphoid cells are removed from the host.
  • Cell ⁇ of lymphoid origin ⁇ uch as splenocytes, lymph node cell ⁇ or other lymphatic tissue or peripheral blend lymphocytes may be obtained from the immunised host.
  • lymphoid cell ⁇ removed are splenocytes, from a mouse, between 2-4 day ⁇ following immuni ⁇ ation. A ⁇ uitable period may be 3-5 day ⁇ .
  • the lymphoid cells are then immortalised.
  • the cell ⁇ from the i muni ⁇ ed ho ⁇ t 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.
  • the incubation lasts for a minimum of 3 days, for example from 4 to 6 days. A suitable period is 5 days.
  • 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 mou ⁇ e. or rat.
  • 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 fusion ⁇ are typically screened by an enzyme-linked immunosorbent assay (ELISA) .
  • ELISA enzyme-linked immunosorbent assay
  • a solid support such as microtitre well ⁇ , i ⁇ coated with adult or fetal human ⁇ AChE.
  • Supernatant from a culture of a fu ⁇ ion i ⁇ 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 i ⁇ generally added after the coated support ha ⁇ been incubated with the culture supernatant and the support has been washed.
  • 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.
  • the labelled antibody i ⁇ incubated with the support in the absence of a PEG provided the ⁇ upernatant i ⁇ 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 ⁇ upernatant, which ha ⁇ bound to the human sAChE coating the solid support.
  • the label is typically an enzyme label such a ⁇ 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:
  • Step (a) may be conducted in vitro in suitable culture media in tissue culture flasks, in a hollow fibre tis ⁇ ue culture device or in a cell fermenter for example.
  • 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.
  • Cell ⁇ of the immortali ⁇ ed cell line may be implanted in a body cavity, such a ⁇ the abdominal cavity, of the animal and allowed to grow.
  • the re ⁇ ulting monoclonal antibody can be separated from the culture medium or from the body cavity fluid such as the a ⁇ cite ⁇ fluid of the animal by technique ⁇ ⁇ uch a ⁇ ammonium sulphate precipitation, ion exchange chromatography, gel filtration, affinity chromatography, high-performance liquid chromatography, etc.
  • the monoclonal antibody can be used in the diagnosi ⁇ of NTD ⁇ and, in particular, anencephaly, encephalocele and spina bifida.
  • the monoclonal antibody can also be u ⁇ ed in the di ' agno ⁇ i ⁇ of omphalocele, ga ⁇ tro ⁇ chi ⁇ i ⁇ , intrauterine death, e ⁇ ophageal atresia, twin pregnancy with acardiac fetus, normal infant, teratoma, ascite ⁇ , cy ⁇ tic hygroma, hypopla ⁇ ia °f heart and lung ⁇ , cloacal ex ⁇ trophy, hydrocele, epidermoly ⁇ i ⁇ bullo ⁇ a dy ⁇ trophica, and apla ⁇ ia cuti ⁇ congenita; a ⁇ well as other neurodegenerative disorder ⁇ ⁇ uch a ⁇ senile dementia, Parkinson' ⁇ disease, and Alzheimer's disease.
  • diagnosi ⁇ depend ⁇ upon detecting and mea ⁇ uring level ⁇ of sAChE.
  • a monoclonal antibody i ⁇ 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 as ⁇ ay may be performed qualitatively, semi-quantitatively or quantitatively.
  • a variety of a ⁇ ay format ⁇ 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 thi ⁇ protein or both to capture and to label thi ⁇ protein.
  • the antibody also may be used in a variety of homogeneous assay format ⁇ in which the protein i ⁇ 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.
  • Thi ⁇ ⁇ urface should be capable of being washed.
  • the type ⁇ of surfaces which may be used include polymer ⁇ of variou ⁇ type ⁇ (moulded into icrotitre well ⁇ ; beads; dipstick ⁇ ; a ⁇ piration tip ⁇ ; electrodes; and optical device ⁇ ) , particle ⁇ (for example latex; stabilized red blood cells (erythrocyte ⁇ ) ; bacterial or fungal cell ⁇ ; spores; gold or other metallic or metal- containing ⁇ ol ⁇ ; organic ⁇ ol ⁇ ; and proteinaceou ⁇ colloid ⁇ ; with the u ⁇ ual 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 po
  • 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; anhydride ⁇ ; amino, hydroxyl, or carboxyl group ⁇ ; sulphydryl groups; carbonyl group ⁇ ; diazo group ⁇ ; un ⁇ aturated groups).
  • Immobilisation may be through a preliminary coating with protein A or antibody to murine immunoglobulin.
  • the captured protein is detected by any means ' which will give a detectable signal.
  • this may be achieved by use of a labelled molecule, in particular antibody, or particle a ⁇ de ⁇ cribed • above which will react with the capture protein.
  • the activity of the immobili ⁇ ed AChE can be a ⁇ ayed.
  • 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 specie ⁇ or fluorophore, or indirectly by labelling the molecule or particle with an enzyme it ⁇ elf capable of giving ri ⁇ e to a measurable change of any ⁇ ort.
  • the detectable ⁇ ignal may be due to, for example, agglutination, diffraction effect or birefringent effect occurring if any of the ⁇ urface ⁇ referred to i ⁇ in the form of particle ⁇ .
  • a preferred assay format is the sandwich assay.
  • a fir ⁇ t antibody capable of binding to fetal human sAChE capture ⁇ 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 i ⁇ 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.
  • the second antibody i ⁇ labelled with an enzyme ⁇ uch a ⁇ an alkaline phosphata ⁇ e or peroxida ⁇ e.
  • a u ⁇ eful ⁇ andwich a ⁇ ay therefore involve ⁇ contacting a te ⁇ t ⁇ ample with enzyme-labelled monoclonal antibody, capturing the re ⁇ ulting immune complex onto a solid surface using a monoclonal or polyclonal antibody capable of binding to human sAChE, removing any exces ⁇ labelled antibody and adding a ⁇ ubstrate for the enzyme.
  • the presence of the human sAChE sample is thu ⁇ revealed.
  • tho ⁇ e ⁇ uitable for immunoa ⁇ say ⁇ including (1) agglutination with the monoclonal antibody ad ⁇ orbed onto particle ⁇ of, for example, poly ⁇ tyrene latex; (2) an enzyme-linked immuno- ad ⁇ orbant a ⁇ ay (ELISA) carried out in microtitre plate ⁇ , (3) a dipstick ELISA with an antibody-coated dip ⁇ tick, and (4) ⁇ andwich a ⁇ ay ⁇ u ⁇ ing ⁇ mall magnetic particle ⁇ coated with capture antibody together with the monoclonal antibody labelled either with coloured particle ⁇ , or particle ⁇ with the potential of colour development, or with an enzyme or a fluorescent moiety.
  • ELISA enzyme-linked immuno- ad ⁇ orbant a ⁇ ay
  • Test kits ⁇ uitable for use in determining human sAChE in a ⁇ ample compri ⁇ e a monoclonal antibody according to the invention and mean ⁇ for determining whether the antibody binds to human ⁇ AChE.
  • Specific components of the kits may be a ⁇ 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-immunoa ⁇ ay typically include ⁇ 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.
  • 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, lOO ⁇ l
  • FsAChE fetal sAChE
  • HRO-labelled goat anti-mou ⁇ e 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 show ⁇ the re ⁇ ult ⁇ of competitive ELISA (CELIA) u ⁇ ing MAB AJ2.
  • CELIA competitive ELISA
  • 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 dilution ⁇ of competing samples. Thi ⁇ was followed with HRO-labelled goat anti-mou ⁇ e Ig conjugate, then, the ⁇ ub ⁇ trate TMB. Re ⁇ ult ⁇ were the average of triplicate mea ⁇ urement ⁇ and are expressed a ⁇
  • Samples were (*) RBC-AChE, DS-AChE, PsChE and NHS.
  • AChE concentration scale refers to FsAChE, AsAChE. RBC AChE and PsChE, while the dilution scale refers to CSF, NHS and Ds-AChE.
  • Figure 3 show ⁇ the re ⁇ ult ⁇ of CELIA u ⁇ ing MAB AJ5 at a dilution of 1:25. Experimental detail ⁇ are a ⁇ for Figure 2.
  • Figure 4 how ⁇ the re ⁇ ult ⁇ of CELIA u ⁇ ing MAB AJ3 at a dilution of 1:50. Experimental detail ⁇ are a ⁇ 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 ⁇ hown. Activities of AChE (m units/ml) in the antigen preparation ⁇ were: fetal brain homogenate-42.6; po ⁇ itive • amniotic fluid-62.2; and CSF-120. Microwell titre plate ⁇ were coated with goat anti-mou ⁇ e ⁇ chain antibodie ⁇ (5 ⁇ g/ml, 100 ⁇ l) and then incubated with MAB AJ2 culture supernantant for 5h at room temperature. After washing, dilutions of ⁇ ample ⁇ were added and incubation continued for 18h at room temperature.
  • Figure 6 shows ⁇ the re ⁇ ult ⁇ of an antibody capture ⁇ andwich ELISA for sAChE u ⁇ ing MAB AJ2. Comparison of binding of maternal serum from pregnancies from a non-NTD fetus (-) and pregnancie ⁇ from a NTD fetus (+) is shown. As ⁇ ay ⁇ were done in triplicate and the standard deviation wa ⁇ ⁇ 10-%. Experimental details are as for Fig 5, but u ⁇ ing the following sample ⁇ : (+) maternal sera A ( ⁇ ) , B (•) . (-) maternal sera F (D) , G (o) . REFERENCE EXAMPLE: Isolation and Partial Characteri ⁇ ation of Human Adult and Fetal sAChE 1. EXPERIMENTAL PROCEDURES
  • Cerebro ⁇ pinal fluid (CSF) and human adult brain were obtained from the Department of Clinical Chemi ⁇ try, South ead Ho ⁇ pital, Bri ⁇ tol, U.K.
  • Human fetal brain ⁇ were di ⁇ ected from 12-18 week old aborted fetu ⁇ e ⁇ . In both ca ⁇ e ⁇ ti ⁇ ue ⁇ were frozen at -70 ° C within Ih of di ⁇ ection.
  • 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 as ⁇ ay ( ⁇ ee below) .
  • the column wa ⁇ calibrated with a Pharmacia High Molecular Weight kit.
  • Enzyme activity i ⁇ expre ⁇ ed a ⁇ IU (micromoles product formed per minute) .
  • Acetylthiocholine iodide (1 mM) was u ⁇ ed a ⁇ ⁇ ubstrate.
  • AChE activity was taken as that activity which wa ⁇ inhibited by l,5-bis-(4-allyldimethyl- ammonium phenyl)-pentane-3-one dibromide (BW 284 C51;
  • Isoelectric focusing (IEF)
  • Analytical isoelectric focusing wa ⁇ carried out at 10*C on 5% polyacrylamide - 0.5% agarose cylindrical gels
  • the gel (100 ⁇ l) wa ⁇ 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) wa ⁇ added in the same buffer (400 ⁇ l) and the su ⁇ pen ⁇ ion wa ⁇ incubated for 4h at 4 C C with gentle mixing.
  • Non- ⁇ pecific binding wa ⁇ determined by u ⁇ ing Agaro ⁇ e-bound goat immuno- globulin G gel (Sigma Co.). Protein Estimation Protein concentration was determined by u ⁇ ing a modified Lowry a ⁇ say (Markwell e_t a_l. , Anal.Biochem..87.206-210. 1978) . Bovine serum albumin was used as a standard.
  • AChE and ChE activities were measured by the Ellman as ⁇ ay u ⁇ ing acetylthiocholine iodine (1 mm) a ⁇ a substrate and BW 284 C51 (1.5 ⁇ m) a ⁇ AChE inhibitor. Protein concentration wa ⁇ estimated by Lowry. Protein concentrations and enzyme activity varied by ⁇ 20% between different preparations.
  • 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.
  • Fraction ⁇ corre ⁇ ponding to each peak were pooled. In the adult brain supernatant, the applied AChE activity was equally divided into the two peak ⁇ , while >70% of the fetal supernatant AChE activity appeared in peak PI.
  • PI contained most of the slow moving AChE (which correspond ⁇ to sAChE in CSF) as well a ⁇ P ⁇ ChE and some of the fast moving AChE.
  • P2 contained some sAChE, a negligible amount of P ⁇ ChE and o ⁇ t of the fa ⁇ t moving AChE.
  • Electrofocu ⁇ ing of purified sAChE wa ⁇ performed using ampholyte ⁇ 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.
  • Table 2 ⁇ how ⁇ that over 80% of both adult and fetal ⁇ AChE wa ⁇ absorbed onto the lectin resins used. Thi ⁇ interaction was specific a ⁇ shown by the lack of binding to Agaro ⁇ e - IgG resin. The binding of ⁇ AChE to the lectin ⁇ was very ⁇ trong and no more than 50% of the bound enzyme could be de ⁇ orbed with the corre ⁇ ponding ligand (0.5M ⁇ -methyl gluco ⁇ ide, 0.5M ⁇ -methyl manno ⁇ ide and 0.25M 3-methyl galacto ⁇ ide) .
  • the corre ⁇ ponding ligand 0.5M ⁇ -methyl gluco ⁇ ide, 0.5M ⁇ -methyl manno ⁇ ide and 0.25M 3-methyl galacto ⁇ ide
  • Rabbit ⁇ were immuni ⁇ ed by intramu ⁇ cular injection with adult sAChE (50 ⁇ g) in Freund's Complete Adjuvant (FCA) . After 4-5 week ⁇ , the rabbit ⁇ were injected similarly and bled 5 day ⁇ later. Antiserum was aliquoted and stored at -20°C.
  • I_n Vivo immunisation A Balb/c mouse wa ⁇ immuni ⁇ ed intraperitoneally with fetal sAChE (50 ⁇ g) in FCA. After 4 weeks, the mouse was injected intravenously with 25 ⁇ g of the antigen and fusion with the myeloma cell ⁇ (X63/Ag8.653) was performed four days later essentially a ⁇ described by Fazekes and Scheidegger (J.Immunol. eth..35.1.1980) .
  • 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 ⁇ M) ) , and thymocyte-conditioned medium (see below) . After 5 day ⁇ in culture the ⁇ plenocyte ⁇ were harve ⁇ ted and fused with X63 myeloma cell ⁇ a ⁇ de ⁇ cribed before.
  • Thymocyte-conditioned medium wa ⁇ prepared a ⁇ follow ⁇ ; Thy u ⁇ glands were di ⁇ sected out of five 12-14 day ⁇ old Balb/c mice.
  • a ⁇ ingle cell suspen ⁇ ion wa ⁇ made and the cell ⁇ were incubated for 3 day ⁇ at 37 ⁇ C at a cell den ⁇ ity of 5,000,000 cell ⁇ /ml in 20% FCS in RPMI1640 medium containing glutamine (2 mM) and 2-mercaptoethanol (50 ⁇ M) .
  • the cell ⁇ were centrifuged and the supernatant (thymocyte-conditioned medium) wa ⁇ filtered (0.22 ⁇ M) and ⁇ tored frozen at -70 ⁇ C.
  • Modified ELISA the a ⁇ ay wa ⁇ performed a ⁇ above but with the addition of polyethyleneglygol (PEG6000) at 4% (w/vol.) to the ⁇ econd antibody step.
  • PEG6000 polyethyleneglygol
  • MAB to ' sAChE were characteri ⁇ ed by u ⁇ ing the following method ⁇ :
  • the reaction wa ⁇ 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) wa ⁇ mixed with variou ⁇ antigen ⁇ olution ⁇ made up to 500 ⁇ l in PBS.
  • Incubation wa ⁇ carried out for 2h at room temperature, then overnight at 4*C.
  • Triton X-100 (0.1% (w/v)) wa ⁇ added to the detergent- ⁇ oluble antigen ⁇ . After incubation, the gel and any bound antigen were spun down at low speed and the gel wa ⁇ wa ⁇ hed twice with PBS. All unbound material wa ⁇ kept for analysis by the Ellman enzyme as ⁇ ay and by PAGE.
  • Enzyme assay for AChE ChE and AChE activity was assayed by the Ellman test.
  • Polyacrylamide gel electrophore ⁇ is wa ⁇ performed a ⁇ de ⁇ cribed by Coupland and Holme ⁇ (Q.J. Micro ⁇ c.Sci. ,98, 327-330) .
  • Microtitre wells were coated overnight with goat anti- mouse IgM antibodies (Sigma) (5 ⁇ g/ml, 100 ⁇ l) overnight at 4 * C. After washing and blocking with assay buffer, 100 ⁇ l 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 anti ⁇ erum in a ⁇ ay buffer wa ⁇ added. After 2h incubation, HRO-labelled goat anti-rabbit IgG wa ⁇ added followed by the substrate.
  • goat anti- mouse IgM antibodies Sigma
  • biotin-labelled rabbit anti-AChE immunoglobulin fraction was added at the appropriate dilution for 2h, followed by horse-radish peroxidase labelled ⁇ treptavidin (Sigma) , then by the substrate.
  • the modified ELISA wa ⁇ used a ⁇ a screening a ⁇ say.
  • 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.
  • 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 wa ⁇ done u ⁇ ing MAB from hybridoma ⁇ that had been cloned three time ⁇ and who ⁇ e antibody content wa ⁇ quite high. Addition of PEG wa ⁇ routinely done to all subsequent as ⁇ ay ⁇ . Fu ⁇ ion experiment (c) produced a number of po ⁇ itive hybrids.
  • Nine of the ⁇ e were cloned three time ⁇ by limiting dilution. Re ⁇ ults obtained with ⁇ ome of the ⁇ e are reported here. All the nine MAB were of the IgM cla ⁇ s.
  • 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 reason ⁇ : (i) to allow multiple use of the gel and (ii) the covalent cross-linkage wa ⁇ found to stabili ⁇ e antibody activity of the MAB.
  • Table 3 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 i ⁇ shown for comparison.
  • Table 4 shows the AChE and pChE enzyme activities of the antigen ⁇ olution ⁇ u ⁇ ed' in the binding studie ⁇ . The ⁇ e activitie ⁇ were determined by the Ellman a ⁇ ay.
  • FIG. 5 show ⁇ titration curve ⁇ for fetal brain homogenate, CSF and a po ⁇ itive (+) AF (AF from an NTD pregnancy) . It can be clearly seen that the as ⁇ ay can detect ⁇ AChE in (+) AF.
  • the Figure al ⁇ o indicate ⁇ that the greate ⁇ t activity i ⁇ found in the CSF and the lea ⁇ t in the fetal brain homogenate.
  • Mea ⁇ urement ⁇ were done in triplicate and the ⁇ tandard deviation wa ⁇ ⁇ 10%. Both (+) and (-) samples showed high absorbance. However, the average absorbance given by the (+) AF samples was markedly higher than that of the (-) AF ⁇ ample ⁇ .
  • Extraction of fetal ⁇ oluble AChE was carried out by thawing the brain ti ⁇ ue and homogeni ⁇ ing in 5 volume ⁇ (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 ⁇ upernatant wa ⁇ removed, aliquoted and ⁇ tored at -70°C until required.
  • the bound material wa ⁇ then eluted with the same buffer containing 12 mM edrophonium chloride (35 ml) .
  • Edrophonium chloride wa ⁇ 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) .
  • Fraction ⁇ containing AChE activity a ⁇ determined by the Ellman assay were pooled, lyophilised, reconstituted in PBS (2 ml) and aliquots frozen at -20°C until required.
  • FCS FCS AChE. or fetal secretary.
  • Splenocytes were removed after 4 day ⁇ and fu ⁇ ed with myeloma cell ⁇ (x63/Ag8.653) e ⁇ sentially as described by Fazekas and Scheidegger (J. Immunol. Meth, 35, 1, 90).
  • Thymus conditioned medium wa ⁇ 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 hr ⁇ or 48 hr ⁇ in Dulbecco ⁇ Modified Eagle ⁇ 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 den ⁇ ity of 5 x 10 6 /ml in 25 cm 2 fla ⁇ k ⁇ . The supernatant wa ⁇ harve ⁇ ted, filtered (0.22 ⁇ m) and stored at -20°C.
  • DMEM Dulbecco ⁇ Modified Eagle ⁇ Medium
  • HT supplement Sigma
  • 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 ⁇ m) and stored at -20°C.
  • 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 ⁇ g/ml fetal sAChE.
  • the medium used wa ⁇ Dulbecco ⁇ Modified Eagles Medium + 5% v/v myoclone fetal calf serum + 33% (v/v) thymus conditioned medium + 25% (v/v) EL4 ⁇ upoplement + 5 x 10 ⁇ 5 M 2-mercaptoethanol + HT ⁇ upplement + 18 mM HEPES pH 7.3. Two 25cm 2 flasks were set up.
  • the cell ⁇ in the ⁇ econd fla ⁇ k were re ⁇ timulated for a further 6 day ⁇ with 0.8 ⁇ g/ml fetal sAChE in fresh medium prior to fu ⁇ ion with myeloma cell ⁇ by the ⁇ ame method.
  • Direct ELISA for screening hvbridoma ⁇ 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 wa ⁇ used at 400 ng/ml. After washing thrice with as ⁇ ay buffer (phosphate buffered saline, pH 7.2, 0.05% Tween-20) , 100 ⁇ l of culture supernatnat wa ⁇ added to each well and the plate incubated for 2 h at 37*C.
  • as ⁇ ay buffer phosphate buffered saline, pH 7.2, 0.05% Tween-20
  • Microtitre wells were coated with human fetal sAChE (400 ng/ml) or Red blood cell AchE (5 ⁇ g/ml) in 50mM sodium carbonate/bicarbonate buffer, pH 9.6, overnight at 4'C 200 ⁇ l/well at all stage ⁇ . After blocking for 30 min, and wa ⁇ hing (2x) with a ⁇ ay buffer (50mM Tri ⁇ /HCl, 0.05% Tween- 20, 0.02% sodium azide, pH 7.4), 200 ⁇ l of each MAB diluted in a ⁇ say buffer plus 0.1% casein wa ⁇ added to each well and the plate incubated for three hour ⁇ at room temperature.
  • a ⁇ ay buffer 50mM Tri ⁇ /HCl, 0.05% Tween- 20, 0.02% sodium azide, pH 7.4
  • Fu ⁇ ion 1 Immunized and boo ⁇ ted in vivo with fetal sAChE coupled to KLH. From an Fl mou ⁇ e. Two cloned hybridoma ⁇ were created. AB 1-1 AB 1-7
  • Fu ⁇ ion 2 Intra ⁇ plenic: Fl mouse. Two cloned hybridomas were created. AB 2-2 AB 2-3.
  • Fu ⁇ ion 3 Intra ⁇ plenic: Fl mou ⁇ e. Three cloned hybridoma ⁇ were created. AB 3-2
  • Fu ⁇ ion 4 Intra ⁇ planic: Fl mou ⁇ e. One cloned hybridoma wa ⁇ created. AB 4-3 AB 4-7.
  • Fu ⁇ ion 5 in vitro immuni ⁇ ation, re ⁇ timulated: Fl mou ⁇ e.
  • Fu ⁇ ion 6 in vitro immuni ⁇ ation, Fl mou ⁇ e. Five cloned hybridoma ⁇ were created. AB 6-2 AB 6-3 AB 6-5 AB 6-7 AB 6-10
  • Fu ⁇ ion 8 Intra ⁇ plenic immuni ⁇ ation, Balb/c mouse. Five cloned hybridoma ⁇ were created:
  • AB 4-7, AB 5-12, AB 3-5 were from ammonium ⁇ ulphate cut ⁇ of crude a ⁇ cite ⁇
  • AB 6-3 wa ⁇ a euglobulin precipitation from crude a ⁇ cite ⁇ .
  • Cells secreting antibody not tested yet or po ⁇ itive a ⁇ cite ⁇ 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.

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Abstract

Un anticorps monoclonal spécifique à l'acétyl cholinestérase secrétoire humaine (AChEs), et ne présentant aucune réactivité croisée avec d'autres types d'acétyl cholinestérase humaine est utile pour déterminer la AChEs humaine dans un échantillon, et par conséquent pour déterminer des anomalies du tube neural telles que l'anencéphalie, l'encéphalocèle et le spina-bifida.
PCT/GB1990/001886 1989-12-04 1990-12-04 Anticorps monoclonaux specifiques a l'acetyl cholinesterase Ceased WO1991008302A1 (fr)

Applications Claiming Priority (2)

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GB898927389A GB8927389D0 (en) 1989-12-04 1989-12-04 Monoclonal antibodies
GB8927389.0 1989-12-04

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WO1991008302A1 true WO1991008302A1 (fr) 1991-06-13

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993005398A1 (fr) * 1991-09-09 1993-03-18 Avalon Biosciences Limited Essai de capture d'enzyme
WO2000073427A3 (fr) * 1999-05-31 2001-02-22 Yissum Res Dev Co Peptides derives de l'acetylcholinesterase et leurs utilisations
EP1270594A1 (fr) * 1996-03-22 2003-01-02 Synaptica Limited Anticorps dirigé contre un fragment soluble de l'acétylcholinestérase
WO2006050667A1 (fr) * 2004-11-12 2006-05-18 Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences ANTICORPS MONOCLONAUX DIRIGES CONTRE L’ACETYLCHOLINESTERASE LIEE A L’APOPTOSE (AR-AChE) ET LEUR USAGE

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOCHEM. SOC. TRANS. vol. 14, no. 6, March 1986, London, GB. pages 1234 - 1235; P. GARDNER et al.: "Isolation and partial characterization of the secretory form of human brain acetylcholinesterase." see the whole document (cited in the application) *
CLIN. CHIM. ACTA vol. 165, no. 1, June 1987, Amsterdam, NL pages 17 - 25; A. RASMUSSEN et al.: "Immunochemical determination of acetylcholinesterase in amniotic fluid. An evaluation of eleven monoclonal antibodies." see page 21, line 10 - page 23, line 15 (cited in the application) *
J. IMMUNOL. METHODS vol. 44, no. 2, 1981, Amsterdam, NL pages 125 - 133; S. COBBOLD et al.: "A rapid solid phase enzyme-linked binding assay for screening monoclonal antibodies to cell surface antigens." see abstract *
NATURE. vol. 256, 1975, LONDON GB & C. MILSTEIN: "Continuous cultures of fused cells secreting antibody to predefined specificity." see the whole document *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993005398A1 (fr) * 1991-09-09 1993-03-18 Avalon Biosciences Limited Essai de capture d'enzyme
EP1270594A1 (fr) * 1996-03-22 2003-01-02 Synaptica Limited Anticorps dirigé contre un fragment soluble de l'acétylcholinestérase
WO2000073427A3 (fr) * 1999-05-31 2001-02-22 Yissum Res Dev Co Peptides derives de l'acetylcholinesterase et leurs utilisations
US7067486B2 (en) 1999-05-31 2006-06-27 Yissum Research Development Company Of The Hebrew University Of Jerusalem Acetylcholinesterase-derived peptides and uses thereof
WO2006050667A1 (fr) * 2004-11-12 2006-05-18 Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences ANTICORPS MONOCLONAUX DIRIGES CONTRE L’ACETYLCHOLINESTERASE LIEE A L’APOPTOSE (AR-AChE) ET LEUR USAGE

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AU6885591A (en) 1991-06-26
GB8927389D0 (en) 1990-01-31

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