WO1993005398A1 - Essai de capture d'enzyme - Google Patents

Essai de capture d'enzyme Download PDF

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Publication number
WO1993005398A1
WO1993005398A1 PCT/GB1992/001650 GB9201650W WO9305398A1 WO 1993005398 A1 WO1993005398 A1 WO 1993005398A1 GB 9201650 W GB9201650 W GB 9201650W WO 9305398 A1 WO9305398 A1 WO 9305398A1
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Prior art keywords
sache
enzyme
ligand
human
antibody
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PCT/GB1992/001650
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English (en)
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David Mark Brennand
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AVALON BIOSCIENCES Ltd
Ortho Clinical Diagnostics Ltd
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AVALON BIOSCIENCES Ltd
Kodak Clinical Diagnostics Ltd
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Priority to CA002118709A priority Critical patent/CA2118709A1/fr
Priority to JP5505072A priority patent/JPH06510851A/ja
Priority to EP92919599A priority patent/EP0604509A1/fr
Publication of WO1993005398A1 publication Critical patent/WO1993005398A1/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • This invention relates to a method and a test kit for determining an enzyme in a sample, especially human secretory acetylcholinesterase.
  • the determination of human secretory acetylcholinesterase enables Neural Tube Defects or Alzheimer's disease to be detected.
  • NTDs Neural Tube Defects
  • CNS central nervous system
  • NTD incidence varies considerably throughout the world. In Europe, the prevalence is 6-13 per 10,000 births.
  • NTDs occur in couples not previously considered at risk from this disorder.
  • Alpha-fetoprotein is a glycoprotein with a molecular weight of 70,000 Daltons. It is usually 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 fetuses.
  • AFP is measured in maternal serum. An elevated value is followed by US scanning, then by
  • amniocentesis The amniotic fluid (AF) is then tested for AFP.
  • AF amniocentesis
  • AFP levels in AF also vary with gestational age. Furthermore, a major problem with assaying AFP in AF is the likelihood of a false positive due to contamination of the AF samples with fetal blood and/or maternal bipod.
  • 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 components are expected to be found in the AF and to diffuse into maternal serum. These could, therefore, be potential markers for NTD.
  • Smith et al. (Lancet, i, 685, 1979) proposed the analysis of AChE in AF as a
  • Cholinesterases are a group of enzymes that hydrolyse
  • AChE acetyl cholinesterases
  • Globular forms are dominant in the central nervous system and red blood cells (RBC). They exist as a monomer, dimer, or tetramer. Globular AChEs are membrane-bound or soluble.
  • AChE In human brain, about 70% of AChE is membrane-bound and 100% of red blood cells AChE is membrane-bound.
  • the central nervous system soluble AChE is in itself of multi- forms. The bulk of it is believed to be derived from the membrane-bound AChE as is shown by its behaviour in the presence of detergents.
  • AChE neural soluble AChE
  • CSF mammalian cerebrospinal fluid
  • AChE inhibitor edrophonium chloride edrophonium chloride
  • a monomer (4.0 S), a dimer (5.5 S), and a tetramer (10.3 S).
  • the origin of the first two is not clear.
  • the dimer is very much similar to the dimer membrane-bound AChE found in red blood cells (RBC).
  • the tetramer form is the neural-specific form, the secretory AChE (sAChE). The amount of this form goes up to 62 -fold in NTD .
  • AChE is currently 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 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 Pedersen et al,
  • AChE from the electropax of Electrophorus electricus has been shown to bind to both arginine- Sepharose as well as edrophonium-Sepharose (Small et al, Neuroscience 21, 991-996, 1987).
  • AChEs purified from some sources possess protease activities (Small et al, 1987 and Small, 1989 Neuroscience. Lett. 95, 307-312, 1989).
  • This work has led to suggestions that some AChEs may be zymogens (i.e. inactive precursors) of proteases (Small, D.H., Neuroscience 29, 241-249, 1989).
  • APP transmembrane amyloid precursor protein
  • APP secretase The protease necessary for this cleavage although previously not identified, has been referred to as "APP secretase”.
  • Biochemistry 30 10795-10799, 1991 purified sAChE from human brain by a method using using an edrophonium-Sepharose column. They called this purified material AChE-associated protease (AChE-AP) and showed that it has the function of the missing "APP secretase”. They showed that the human AChE-AP so purified cleaved the ⁇ A4 protein sequence
  • Alzheimer's disease loss of sAChE activity or a deficiency in levels of the enzyme may result in localised loss of.
  • cerebrospinal fluid of patients with Alzheimer's disease (Navaratnam, D.S., Priddle, J.D., McDonald B., Esiri, M.M., Robinson, J.R., and Smith, A.D. Lancet 337, 447-450, 1991.
  • acetylcholinesterase in a test sample, which method comprises contacting a ligand which is capable of binding sAChE and which is not an antibody with the sample and determining whether human sAChE has bound to the ligand.
  • ligand which is immobilised on the surface of a solid or matrix support, which ligand is:
  • This may be a test to, for example, measure antibody in serum or monoclonal antibody secreted by a cell line.
  • the ligand in this test method may be, for example, any of the ligands used in the previous methods.
  • antibody in the above methods includes immunoreactive antibody fragments such as Fab fragments.
  • An enzyme capture assay can be used to measure levels of an enzyme, especially human sAChE, in biological fluids and other samples.
  • the assay includes, as its first step, the coupling or binding onto a solid or matrix support of the ligand to which the enzyme, e.g. sAChE, is able to actively bind. This enables the enzyme to be actively bound from a solution, or biological fluid.
  • a detection stage i.e. via an antibody capable of binding to the enzyme, for an Enzyme Capture ELISA
  • an assay is produced which is able to measure quantitatively levels of the enzyme, e.g. sAChE, in a sample, solution or biological fluid.
  • the method according to the present invention when applied to sAChE can be used in the diagnosis of NTDs and, in particular, anencephaly, encephalocele and Spina bifida.
  • the method can also be used in the diagnosis of omphalocele, gastroschisis, intrauterine death, esophageal atresia, twin pregnancy with acardiac fetus, normal infant, teratoma, ascites, cystic hygroma, hypoplasia of heart and lungs, cloacal exstrophy, hydrocele, epidermolysis bullosa
  • dystrophica and aplasia cutis congenita: as well as other neurodegenerative disorders such as senile dementia,
  • Parkinson's disease and Alzheimer's disease.
  • the method may also be used in the diagnosis of Down's Syndrome. In each case, diagnosis may depend upon detecting and measuring levels of sAChE. For the diagnosis of these conditions the ligand must bind AChE either the foetal form or the adult form or both.
  • the method of the present invention may, for example, be used to diagnose Neural Tube Defects in a fetus.
  • the presence of Neural Tube Defects is indicated by, for example, increased sAChE in the amniotic fluid, maternal serum or cerebrospinal fluid.
  • the method may also, for example, be used to diagnose Alzheimer's disease in an adult.
  • the presence of Alzheimer's disease is indicated by, for example, decreased sAChE in human serum and abnormal levels of sAChE in cerebrospinal fluid.
  • the present method can be used as a screen to be used while raising monoclonal antibodies specific for the enzyme determined, e.g. human sAChE.
  • An enzyme such as adult or fetal human sAChE can therefore be assayed using the present invention.
  • a sample suspected of containing the enzyme is contacted with the ligand.
  • the sample may be a biological fluid such as cerebrospinal fluid, or amniotic fluid or human serum.
  • the sample may be diluted in a buffer such as phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • a detergent may be present such as Tween.
  • the sample may be mixed with high ionic strength buffer in order to increase the ability of the assay to distinguish between samples containing raised levels of the enzyme, e.g. sAChE, and normal samples.
  • the sample may therefore be mixed with a solution having a higher ionic strength than that of physiological saline.
  • conductivity of the solution may be greater than that of physiological saline.
  • PBS having an ionic strength at least as great as that of 0.15M NaCl may be used.
  • the ionic strength may be that of 0.15 to 1M, preferably 0.15 to 0.5M and more preferably from 0.2 to 0.4M, NaCl. If a Tris
  • the buffer is at least 25mM Tris buffer.
  • An assay may be performed qualitatively, semiquantitatively or quantitatively. A variety of assays
  • the ligand can be used to capture the enzyme, e.g. adult or fetal human sAChE, from solution selectively onto a surface separable from the solution such as a solid surface or matrix to label selectively this enzyme or both to capture and to label this protein.
  • the ligand also may be used in a variety of homogeneous assay formats in which the enzyme is detected in solution with no separation of phases.
  • the types of assay in which the ligand is used to capture the enzyme from solution involve immobilization of the ligand onto a surface. This surface should be capable of being washed.
  • the types of surfaces which may be used include polymers of various types (moulded into Microtiter (trade mark) wells; beads; dipsticks; aspiration tips;
  • a capillary fill device carbon discs (for an electrochemical enzyme immunoassay); particles (for example latex; stabilized red blood cells (erythrocytes); bacterial or fungal cells; star-burst dendrimers; spores; gold or other metallic or metalcontaining sols; organic sols; and proteinaceous colloids; with the usual 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).
  • particles for example latex; stabilized red blood cells (erythrocytes); bacterial or fungal cells; star-burst dendrimers; spores; gold or other metallic or metalcontaining sols; organic sols; and proteinaceous colloids; with the usual size of the particle being from 0.005
  • the attachment of the ligand to the surfaces can be by passive adsorption from a solution 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 include acid halides; anhydrides; amino, hydroxyl, or carboxyl groups; sulphydryl groups; carbonyl groups; diazo groups; epoxy groups; unsaturated groups).
  • the ligand used for binding to the enzyme in the general assay method of the invention is a substrate or substrate analogue, an effector (e.g. an inhibitor
  • analogue (competitive or non-competitive) or activator of, for example, competitive, allostearic or isostearic sites) or effector analogue, a co-factor or co-factor analogue, a coenzyme or co-enzyme analogue or a prosthetic group or prosthetic group analogue of the enzyme.
  • the analogues may be, for example, synthetic analogues.
  • the ligand which can be used to determine human sAChE or antibodies to sAChE is typically a positively charged compound, or a positively chargeable compound, capable of binding to an anionic locus of human sAChE.
  • the ligand may, for example, be a substrate, effector,
  • the ligand may comprise an amino acid such as arginine, histidine, lysine or ornithine which is capable of being positively charged.
  • the ligand may comprise two or more such amino acids, for example as a dimer or other polymer.
  • the amino acid may be in D- or L- form.
  • a useful ligand is a polymer, typically a homopolymer, of an amino acid capable of being positively charged.
  • a ligand may therefore be selected from
  • a mixture of two or more such ligands may be employed.
  • Such polyamino acids may have a molecular weight of from 350 to 1,000,000, for example 4,000 to 300,000. Polyamino acids adsorb easily onto plastic surfaces to facilitate presentation of the ligand. Further, multiple copies of the ligand (i.e. the amino acid) are simultaneously presented for sAChE to bind to.
  • the captured enzyme is detected by any means which will give a detectable signal.
  • a detectable signal For example, this may be achieved by use of a molecule, in particular monoclonal or polyclonal antibody, or particle as described above which will react with the captured enzyme.
  • the antibody is capable of binding to, and typically is specific for the enzyme, e.g. human sAChE.
  • the molecule or particle may be labelled or may be capable of being labelled.
  • a first unlabelled antibody capable of binding to the enzyme e.g. human sAChE
  • the activity of the immobilised enzyme e.g. AChE, can be assayed.
  • the monoclonal antibody may be a monoclonal antibody which is specific for the enzyme but which shows no cross-reactivity with other types of enzyme, for example one which is specific for human sAChE but which shows no crossreactivity with other types of human AChE according to WO 91/08302.
  • the monoclonal antibody may therefore show no reactivity with human red blood cell AChE, and preferably no cross-reactivity with human serum AChE, human membrane-bound neuronal AChE, human muscle AChE and neuronal-soluble non- secretory AChE.
  • the antibody does not cross-react either with human serum pseudo-ChE.
  • the monoclonal antibody may be specific for fetal human sAChE or adult human sAChE.
  • the monoclonal antibody may be IgA, IgD, IgE, IgG or IgM. It may be a monoclonal antibody of any animal species, for example a mammalian monoclonal antibody such as a rat, mouse or human monoclonal antibody.
  • a polyclonal antibody may be a monoclonal antibody of any animal species, for example a mammalian monoclonal antibody such as a rat, mouse or human monoclonal antibody.
  • polyclonal antibody may be raised in an appropriate animal, typically mammal, for example mouse, rat, sheep, goat or rabbit.
  • the detectable signal may be optical or radioactive 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 species or fluorophore, or indirectly by labelling the molecule or particle with an enzyme itself capable of giving rise to a measurable change of any sort.
  • the detectable signal may be due to, for example, agglutination
  • a preferred assay format is the sandwich assay.
  • a test sample may be brought into contact with a ligand which is capable of binding to the enzyme, e.g. fetal human sAChE, and which is immobilised on a support separable from the sample, for example on a solid support.
  • the ligand captures the enzyme in the test sample onto the support and an antibody capable of binding to the enzyme labels the captured enzyme.
  • the antibody may be a monoclonal antibody or a polyclonal antibody.
  • the capturing and labelling operations may be performed in any order or simultaneously.
  • the antibody may be labelled with an enzyme such as an alkaline phosphatase or peroxidase or ⁇ -galactosidase.
  • a useful sandwich assay comprises:
  • Another useful sandwich assay format involves contacting a test sample with enzyme-labelled antibody, capturing the resulting immune complex onto a solid surface using a ligand capable of binding to the enzyme, e.g. human sAChE, removing any excess labelled antibody and adding a substrate for the enzyme label in the enzyme-labelled antibody. The presence of the enzyme, e.g. the human sAChE sample, is thus revealed.
  • enzyme-labelled antibody e.g. human sAChE
  • polystyrene latex (2) an enzyme-linked immuno-adsorbant assay (ELISA) carried out in microtitre plates, (3) a dipstick ELISA with a ligand-coated dipstick, and (4) sandwich assays using small magnetic particles coated with ligand together with the antibody or ligand labelled either with coloured particles, or particles with the potential of colour development, or with an enzyme or a fluorescent moiety.
  • ELISA enzyme-linked immuno-adsorbant assay
  • Test kits suitable for use in determining an enzyme, e.g. human sAChE, in a sample comprise the ligand as defined above and means for detecting the enzymes, e.g.
  • kits for determining whether the ligand, in use, binds to the enzyme, e.g. human sAChE.
  • Specific components of the kits may be as described above.
  • the kits may also comprise one or more additional components including one or more control, one or more buffer and one or more diluent. A standard curve may be provided.
  • a kit for use in an enzyme-immunoassay typically includes an enzyme-labelled reagent and a substrate for the enzyme label.
  • the enzyme label may either be bound to ligand which can bind to captured enzyme, e.g. human sAChE, or be bound to polyclonal or monoclonal antibody capable of binding to the captured enzyme, e.g. human sAChE.
  • the means for detecting enzyme, e.g. human sAChE, bound to the ligand may comprise an enzyme-labelled antibody which is capable of binding to the enzyme, e.g. human sAChE, and a substrate for the enzyme label in the enzyme-labelled antibody.
  • the means for detecting the enzyme e.g.
  • human sAChE bound to the ligand may comprise a first unlabelled antibody which is capable of binding to the enzyme, e.g. human sAChE, a second enzyme-labelled antibody which is capable of binding to the first antibody and a substrate for the enzyme label in the second enzyme-labelled antibody.
  • Figure 1 shows the results of the Enzyme Capture ELISA of Example 4 in which concentration of added sodium chloride (M) (x-axis) is plotted against optical density at 405nm, ⁇ denotes cerebrospinal fluid (CSF), ⁇ denotes spina bifida serum A, * denotes anencephalic serum C, ⁇ denotes normal serum E, ⁇ denotes normal serum D and ⁇ denotes lysed normal serum.
  • M cerebrospinal fluid
  • Figure 2 shows a typical cerebrospinal fluid reference curve employed in Example 6 in which sAChE units (x-axis) are plotted against optical density at 405nm.
  • Figure 4 shows sAChE (upper line) and AFP (lower line) results in NTD cases expressed as multiples of the median, ⁇ denoting anencephaly and ⁇ denoting spina bifida.
  • Figure 5 shows sAChE levels in units for NTD pregnancies relative to the upper limit of normal (mean + twice the standard deviation, or 97.5 centile) of equal to 43.5 units, the symbols being the same as for Figure 4.
  • Cerebrospinal fluid (CSF) and human adult brain were obtained from the Department of Clinical Chemistry, Southmead Hospital, Bristol, U.K. Human fetal brains were dissected from 12-18 week old aborted fetuses. In both cases tissues were frozen at -70°C within lh of dissection.
  • Extraction of soluble AChE was carried out by thawing the brain tissue 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°C. The supernatant was removed, aliquoted and stored at -70°C until required.
  • Cholinesterase activity was measured at 30°C by the method of Ellman et al. (Biochem.Pharm.,7,88-95, 1961). Enzyme activity is expressed as IU (micromoles product formed per minute). Acetylthiocholine iodide (1 mM) was used as substrate. AChE activity was taken as that activity which was inhibited by 1,5-bis-(4-allyldimethyl-ammonium phenyl)-pentane-3-one dibromide (BW 284 C51;
  • AChE activity in different samples was located on 6% polyacrylamide slab gels (8 cm x 8 cm) by the
  • the sedimentation coefficient of sAChE (300 ⁇ l) was estimated in 4-20% (w/v) sucrose gradients (5 ml) in
  • the pi of the enzyme was determined from a standard plot constructed by electrofocusing a number of marker proteins of known pi (IEF calibration kit, Sigma Co.)
  • Protein concentration was determined by using a modified Lowry assay (Markwell et al., Anal.Biochem., 87, 206- 210, 1978). Bovine serum albumin was used as a standard. 2. RESULTS
  • AChE and ChE activities were measured by the Ellman assay using acetylthiocholine iodine (1 mm) as a substrate and BW 284 C51 (1.5 ⁇ m) as AChE inhibitor.
  • Brain supernatant was fractionated by gel filtration on a Sephacryl S-200 column. Three major protein peaks were obtained, eluting at molecular weights, ⁇ 250,000 (void volume), 50,000 - 60,000 and ⁇ 10,000, respectively.
  • edrophonium chloride-eluted fraction by PAGE showed the presence of one band of enzyme activity which was completely inhibited by BW 284 C51 and had an electro-phoretic mobility corresponding 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 position 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.
  • Table 2 A summary of the activities of AChE and PsChE in various human biological samples is shown in Table 2. This Table also shows the ability of edrophonium-Sepharose to bind to the various AChE preparations. Table 2
  • Electrofocusing of purified sAChE was performed using ampholytes 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 3 shows that over 80% of both adult arid fetal sAChE was absorbed onto the lectin resins used. This interaction was specific as shown by the lack of binding to Agarose - IgG resin. The binding of sAChE to the lectins was very strong and no more than 50% of the bound enzyme could be desorbed with the corresponding ligand (0.5M ⁇ -methyl glucoside, 0.5M ⁇ -methyl mannoside and 0.25M ⁇ -methyl galactoside).
  • Microtiter plates were "Immunoplate II" from Gibco (U.K.) Monoclonal anti-Rabbit Immunoglobulins Alkaline Phosphatase Conjugate Clone RG-16 was from Sigma Chemical Co. Ltd.
  • Casein (light white soluble) was from B.D.H..
  • a rabbit (Rb35) was immunised by intramuscular injection with sAChE (50 ⁇ g) in Freunds Complete Adjuvant (FCA). After 4 weeks, the rabbit was boosted by injection with sAChE (50 ⁇ g) in Freunds
  • Normal rabbit serum was from an unimmunised rabbit.
  • Tween 20 polyoxyethylenesorbitan monolaurate
  • Microtiter wells were coated overnight at room temperature with sAChE (500ng/ml) in 0.05M bicarbonate/carbonate buffer pH 9.6. Wells were next washed with PBS/Tween a total of three times, the final wash being left at 37°C for 30 minutes. Rabbit antisera, at a dilution of 1/1000 in
  • PBS/Tween/1% casein were incubated in wells for 2 hours at 37°C. Wells were washed five times with PBS/Tween.
  • bicarbonate/carbonate buffer pH 9.6 was incubated for 30 minutes.
  • the substrate reaction was stopped by the addition of 50 ⁇ l of 1M sodium hydroxide, and optical density readings were measured at 405nm using a Titertek Multiscan MCC plate reader.
  • MATERIALS Microtiter plates were "Immunoplate II" from Gibco (U.K.) Poly amino acids were from Sigma Chemical Co. Ltd. (U.K.) Monoclonal anti-Rabbit Immunoglobulins Alkaline Phosphatase Conjugate Clone RG-16 was from Sigma Chemicals Co. Ltd.
  • Casein (light white-soluble) was from B.D.H..
  • Spina bifida serum A was a pool of 4 sera, from source B1, in equal ratios from mothers carrying Spina bifida positive foetuses.
  • Spina bifida serum B was a pool of 5 sera, from source S1, in equal ratios from mothers carrying Spina bifida positive foetuses.
  • Anencephalic serum C was a pool of 5 sera, from source S1, in equal ratios from mothers carrying Anencephalic positive foetuses.
  • Normal human sera D and E were pools of 5 sera each, from source S3, in equal ratios from a normal pool of pregnant mothers.
  • CSF was a pool of 20 CSF samples, from source S2, in equal ratios.
  • Tween 20 polyoxyethylenesorbitan monolaurate
  • Microtiter wells were coated overnight at 4°C with poly-L- lysine solution (50 ⁇ g/ml poly-L-lysine, Molecular weight
  • Substrate solution (p-nitrophenyl phosphate 2mg/ml in 0.05M bicarbonate/carbonate buffer pH 9.6) was incubated for 6 minutes. The substrate reaction was stopped by the addition of 50 ⁇ l of IM sodium hydroxide, and optical density readings were measured at 405nm using a Titertek Multiscan MCC plate reader.
  • An Enzyme Capture ELISA was set up using poly-L-lysine as capturing agent and rabbit anti-sAChE antisera as the detecting agent.
  • Table 5 shows that where no poly-L-lysine was used to coat the microtiter plate wells, resulting optical density readings at the end of the assay were negligible. This shows that in such a case wells did not have the ability to specifically adsorb the sAChE from the samples, and therefore no detection was observed. Where poly-L-lysine was used to coat the wells significant positive signals were obtained for the three disease
  • Tween 20 polyoxyethylenesorbitan monolaurate
  • Microtiter wells were coated overnight at 4°C with poly-L- lysine solution (50 ⁇ g/ml poly-L-lysine, 150,000-300,000, in PBS). Wells were next washed with PBS a total of three times and blocked with PBS/Tween at 37°C for 30 minutes. Wells were washed with PBS three times. Human serum samples or cerebrospinal fluid (CSF), at an appropriate dilution in one of four solutions PBS, PBS/Tween, PBS plus 0.35M sodium chloride or PBS/Tween plus 0.35M sodium chloride) were incubated in wells for 2 hours at 37°C. Wells were washed five times with PBS. Rabbit anti-sAChE serum at a dilution of 1/1000 in PBS/1% casein was incubated in wells for 110 minutes at 37°C. Wells were washed five time with PBS.
  • poly-L- lysine solution 50 ⁇ g/ml poly-
  • bicarbonate/carbonate buffer pH 9.6 was incubated for 5 minutes.
  • the substrate reaction was stopped by the addition of 50 ⁇ l of 1M sodium hydroxide, and optical density readings were measured at 405nm using a Titertek Multiscan MCC plate reader.
  • An Enzyme Capture ELISA was set up using poly-L-lysine as capturing agent and rabbit anti-sAChE antisera as the detecting agent.
  • PBS/Tween/0.35M sodium chloride appears to be the best solution in which to load the sample as it gives negligible optical density signals for normal sera whereas CSF and sera from mothers carrying foetuses with either Spina bifida or Anencephaly show positive signals.
  • Tween 20 polyoxyethylenesorbitan monolaurate
  • Microtiter wells were coated overnight at 4°C with poly-L- lysine solution (50 ⁇ g/ml poly-L-lysine, 150,000-300,000, in PBS). Wells were next washed with PBS a total of three times and blocked at 37°C for 30 minutes with PBS/Tween. Wells were washed five times with PBS/Tween. CSF at a dilution of 1/6 in PBS/Tween/0.35M sodium chloride was used at the sample stage as the source of sAChE and was incubated in all wells for 2 hours at 37°C. Wells were washed five times with PBS/Tween.
  • An Enzyme Capture ELISA was set up using poly-L-lysine as capturing agent and rabbit anti-sAChE antisera as the detecting agent.
  • Example 1 Described in Example 1.
  • Lysed normal human serum was from a nonpregnant woman.
  • Tween 20 polyoxyethylenesorbitan monolaurate
  • Microtiter wells were coated for 48 hours at 4°C with poly- L-lysine solution (50 ⁇ g/ml poly-L-lysine, 150,000-300,000, in PBS). Wells were next washed with PBS-Tween a total of three times, the final wash being left at 37°C for 30 minutes. Two more washes with PBS/Tween were then
  • CSF cerebrospinal fluid
  • Substrate solution (p-nitrophenyl phosphate 2mg/ml in 0.05M bicarbonate/carbonate buffer pH 9.6) was incubated for 15 minutes. The substrate reaction was stopped by the addition of 50 ⁇ l of 1M sodium hydroxide, and optical density readings were measured at 405nm using a Titertek Multiscan MCC plate reader.
  • An Enzyme Capture ELISA was set up using poly-L-lysine as capturing agent and rabbit anti-sAChE antisera as the detecting agent.
  • Poly-L-lysine was of molecular weight 150,000-300,000
  • Poly-D-lysine was of molecular weight 150,000-300,000.
  • Poly-L-asparagine was of molecular weight 5,000-15,000.
  • Poly-L-glutamate was of molecular weight 50,000-100,000.
  • Poly-L-histidine was of molecular weight 15,000-50,000.
  • Poly-L-ornithine was of molecular weight 100,000-200,000.
  • Poly-L-arginine was of molecular weight 70,000-150,000.
  • Poly-L-aspartate was of molecular weight 15,000-50,000.
  • rabbit anti-sAChE was as described in Reference Example 2.
  • the CSF used in the study was produced by pooling 20 CSF samples obtained from Wessex Regional Immunology,
  • Tween 20 polyoxyethylenesorbitan monolaurate
  • Microtiter wells were coated overnight at 4°C with polyamino acid solutions (50 ⁇ g/ml poly-amino acid in PBS).
  • PBS/Tween/1%casein was incubated in wells for 2 hours at 37°C. Wells were washed five times with PBS/Tween.
  • Monoclonal anti-rabbit immunoglobulin - alkaline phosphatase conjugate at a dilution of 1/1000 in PBS/Tween/l%casein was incubated in wells for 1.5 hours at 37°C. Wells were washed five times with PBS/Tween.
  • Substrate solution p- nitrophenyl phosphate 2mg/ml in 0.05M bicarbonate/carbonate buffer pH 9.6 was incubated for 15 minutes. The substrate reaction was stopped by the addition of 50 ⁇ l of 1M sodium hydroxide, and optical density readings were measured at 405nm using a Titertek Multiscan MCC plate reader.
  • poly-amino acids as capturing agents, and rabbit anti-sAChE antisera as the detecting agent.
  • the results are presented in Table 8. From Table 8 it can be seen that poly-L-lysine (300K), poly-L-lysine (15K), poly-D-lysine, poly-L- histidine, poly-L-ornithine, and poly-L-arginine are all able to act as ligands for sAChE to bind to giving positive optical density readings in the Enzyme Capture ELISA.
  • Poly- L-asparagine, poly-L-glutamate, and poly-L-aspartate only give final optical density values equating with those where PBS was used in place of the poly- amino acids (i.e. control wells), and were therefore unable to be bound by the sAChE.
  • rabbit anti-sAChE was as described in Example 1.
  • the CSF standard used in the study was produced by pooling 20 CSF samples obtained from Wessex Regional Immunology, Southampton General Hospital.
  • Tween 20 polyoxyethylene sorbitan monolaurate
  • Microtiter wells were coated overnight at 4°C with poly-L- lysine solution (50 ⁇ g/ml poly-L-lysine, 150,000-300,000, in PBS). Wells were next washed with PBS/Tween, a total of five times, the final wash being left at 37°C for 1 hour.
  • PBS/Tween/l%casein was incubated in wells for 2 hours at 37°C. Wells were washed five times with PBS/Tween.
  • Monoclonal anti-rabbit immunoglobulin-alkaline phosphatase conjugate at a dilution of 1/1000 in PBS/Tween/l%casein was incubated in wells for 1.5 hours at 37°C. Wells were washed five times with PBS/Tween.
  • Substrate solution p- nitrophenyl phosphate 2mg/ml in 0.05M bicarbonate/carbonate buffer pH 9.6 was incubated for 15 minutes. The substrate reaction was stopped by the addition of 50 ⁇ l of 1M sodium hydroxide, and optical density readings were measured at 405nm using a Titertek Multiscan MCC plate reader. Expression of results
  • Levels of sAChE determined by the Enzyme Capture ELISA are expressed in arbitrary units relative to the standard curve reference CSF performed in each case. The units are defined thus:
  • any serum which had a value greater than 100 Units was reassayed on a plate where CSF at 1/3 dilution (i.e. 200 Units) was the highest reference value.
  • a typical CSF reference standard curve is shown in Fig. 2.
  • Figure 3 showed a relatively constant mean from 15-20 weeks gestation.
  • the five results for week 14 were significantly higher than week 15-20 levels and those levels were not included in the overall reference range.
  • a total of 21 sera were analysed, consisting of 4 from cases of anencephaly and 17 from cases of open spina bifida.
  • Microtiter plates were "Immunoplate II" from Gibco (UK).
  • Poly-L-lysine hydrobromide cat. no. P-1399 molecular weight 289,000 was from Sigma Chemical
  • Tween 20 polyoxyethylenesorbitan monolaurate
  • Tween 20 polyoxyethylenesorbitan monolaurate
  • poly-L-lysine solution 50 ⁇ g/ml poly-L-lysine in PBS.
  • PBS/Tween/11 ⁇ 2 casein was incubated in wells for 1% hours at 37°C. Wells were washed five times with PBS/Tween.
  • bicarbonate/carbonate buffer pH 9.6 was incubated for 1 hour at room temperature.
  • the substrate reaction was stopped by the addition of 50 ⁇ l of 1M sodium hydroxide, and optical density readings were measured at 405nm using a Titertek Multiscan MCC plate reader.
  • Example 6 As per Example 6. The CSF had been frozen and thawed a further three times and had been stored for a further 9 months following its use in Example 6. Therefore the activity had dropped and the defined units from this Example cannot be compared directly with those of the standard curve from Example 6. The arbitrary unit definition used was however the same as for Example 6. Results
  • the efficiency of the criteria used in the diagnosis was estimated to be 80% (personal communication by Dr R Jones RICE).
  • One putative Alzheimer's disease sample gave a value of 70.5 sAChE units, a value which was vastly higher than for the other patients. This value alters the mean for patients from 25.9 sAChE units to 30.88 sAChE units. This patient is the only one who was on antidepressant drugs at the time of taking of sera.

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Abstract

Procédé de dosage de l'acétycholinestérase sécrétoire humaine (AChEs) dans un prélèvement d'essai, lequel procédé consiste à mettre en contact un ligand, qui est capable de se lier à l'AChEs et qui n'est pas un anticorps, avec le prélèvement et à établir si l'AChEs humaine est liée au ligand. L'invention concerne également un procédé de dosage d'un enzyme dans un prélèvement d'essai consistant i) à mettre en contact le prélèvement avec un ligand qui est immobilisé sur la surface d'un support solide ou matriciel, lequel ligand est a) un substrat ou un substrat analogue de l'enzyme; b) un effecteur ou un effecteur analogue de l'enzyme; c) un co-facteur ou un co-facteur analogue de l'enzyme; d) un co-enzyme ou un co-enzyme analogue de l'enzyme; ou e) un groupe prosthétique ou un groupe prosthétique analogue de l'enzyme; et ii) à établir si l'enzyme est lié au ligand.
PCT/GB1992/001650 1991-09-09 1992-09-09 Essai de capture d'enzyme Ceased WO1993005398A1 (fr)

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CA002118709A CA2118709A1 (fr) 1991-09-09 1992-09-09 Essai par capture enzymatique
JP5505072A JPH06510851A (ja) 1991-09-09 1992-09-09 酵素捕獲アッセイ
EP92919599A EP0604509A1 (fr) 1991-09-09 1992-09-09 Essai de capture d'enzyme

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GB919119240A GB9119240D0 (en) 1991-09-09 1991-09-09 Enzyme capture assay for human secretory acetyl cholinesterase
GB9119240.1 1991-09-09

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008543296A (ja) * 2005-06-14 2008-12-04 セルゾーム・アクチェンゲゼルシャフト 酵素と相互作用する新規化合物の同定法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0124103A2 (fr) * 1983-04-29 1984-11-07 BioWhittaker, Inc. Essai fluorométrique de chymopapaine et réactifs pour sa mise en oeuvre
EP0206200A2 (fr) * 1985-06-18 1986-12-30 Yeda Research And Development Company Limited Protéines d'origine humaine du type cholinestérase et leur production
WO1990003577A1 (fr) * 1988-09-30 1990-04-05 The University Of Vermont And State Agricultural College Immunoanalyses pour la detection de proteases de serine presentant une activite catalytique
WO1991008302A1 (fr) * 1989-12-04 1991-06-13 Avalon Biosciences Limited Anticorps monoclonaux specifiques a l'acetyl cholinesterase

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0124103A2 (fr) * 1983-04-29 1984-11-07 BioWhittaker, Inc. Essai fluorométrique de chymopapaine et réactifs pour sa mise en oeuvre
EP0206200A2 (fr) * 1985-06-18 1986-12-30 Yeda Research And Development Company Limited Protéines d'origine humaine du type cholinestérase et leur production
WO1990003577A1 (fr) * 1988-09-30 1990-04-05 The University Of Vermont And State Agricultural College Immunoanalyses pour la detection de proteases de serine presentant une activite catalytique
WO1991008302A1 (fr) * 1989-12-04 1991-06-13 Avalon Biosciences Limited Anticorps monoclonaux specifiques a l'acetyl cholinesterase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY vol. 30, no. 44, 1991, EASTON, PA US pages 10795 - 10799 D. H. SMALL ET AL. 'A protease activity associated with acetylcholinesterase releases the membrane-bound form of the amyloid protein precursor of alzheimer's disease' cited in the application *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008543296A (ja) * 2005-06-14 2008-12-04 セルゾーム・アクチェンゲゼルシャフト 酵素と相互作用する新規化合物の同定法

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CA2118709A1 (fr) 1993-03-18

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