EP2457093A1 - Procédé, notamment un dosage par la méthode elisa, pour détecter in vitro des auto-anticorps bêta amyloïde, plaque de microtitration et kit d'essai - Google Patents

Procédé, notamment un dosage par la méthode elisa, pour détecter in vitro des auto-anticorps bêta amyloïde, plaque de microtitration et kit d'essai

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Publication number
EP2457093A1
EP2457093A1 EP10747569A EP10747569A EP2457093A1 EP 2457093 A1 EP2457093 A1 EP 2457093A1 EP 10747569 A EP10747569 A EP 10747569A EP 10747569 A EP10747569 A EP 10747569A EP 2457093 A1 EP2457093 A1 EP 2457093A1
Authority
EP
European Patent Office
Prior art keywords
microtiter plate
solution
solid phase
antigen
wells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP10747569A
Other languages
German (de)
English (en)
Inventor
Michael Bacher
Richard Dodel
Karthikeyan Balakrishnan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BALAKRISHNAN, KARTHIKEYAN
Original Assignee
Philipps Universitaet Marburg
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Philipps Universitaet Marburg filed Critical Philipps Universitaet Marburg
Publication of EP2457093A1 publication Critical patent/EP2457093A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • 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
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • 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

  • the invention relates to a method, in particular an enzyme-linked immunosorbent assay (ELISA), for in vitro detection of amyloid beta autoantibodies according to the preamble of claim 1, a microtiter plate according to claim 17, and a test kit according to claim 23.
  • ELISA enzyme-linked immunosorbent assay
  • Analyte an antigen to be detected in a sample to be examined or an antibody to be detected.
  • Antigen A protein / polypeptide that causes the production of
  • Antibodies causes when it is injected into an animal organism (antigen stimulus).
  • Antibody A protein that is produced in response to the antigen stimulus and specifically recognizes and binds the stimulus-producing antigen.
  • anti-species antibody An antibody that is produced when proteins (including
  • Antibodies of a species of another species, recognize and bind to all antigens derived from the first species.
  • Binding partner An antigen or antibody that specifically binds to the analyte.
  • Detection antibody binds to the analyte, but not to the binding partner of the analyte and is either with a detection agent or with an immediately detectable substance, such as a
  • the detection means may be an enzyme which is capable of cleaving a specific substrate such that a color reaction is produced.
  • the detection antibody may also be referred to as
  • the course of such a detection method is basically as follows: The binding partner of the analyte is immobilized on a solid phase. Subsequently, the solid phase is incubated with a sample possibly containing the analyte to be detected. If analyte is present in the sample, it binds to the immobilized on the solid phase binding partner and is also immobilized in this way. The binding of the analyte to its binding partner is then detected by means of a detection antibody. For this purpose, the solid phase, on which now the binding partner holds the analyte, incubated with a solution containing the detection antibody. This binds to the immobilized analyte and can then be detected, for example, by carrying out an enzyme-substrate reaction with the aid of the enzyme conjugated to the detection antibody.
  • Such detection methods are preferably used in medical diagnostics in order to obtain tissue or body fluid samples from animals and / or humans Presence or absence of antigens. From the result of the proof then for example statements about possible illnesses of the examinee can be made.
  • a prominent example of diseases that can be examined in this way is Alzheimer's disease. This disease is characterized by a number of neuropathological characteristics, one of which is particularly typical of the formation of so-called neuritic plaques in the brain of patients. These plaques are composed of extracellularly deposited amyloid beta-peptides (A ⁇ ) that result from the cleavage of the amyloid precursor protein (APP) (Kang et al., Nature, 1987.
  • a problem with conventional ELISA for detecting A ⁇ is that not all A ⁇ forms are actually pathological, and that antibodies that specifically recognize the pathological forms are only available to a very limited extent and therefore are very expensive, so at least two specific antibodies are required for detection - one as an immobilized binding partner on the solid phase and a second as a detection antibody Accordingly, also come from different species, since the detection otherwise the risk of a very high background The risk of delivering a high number of false positive results Although the formation of nonspecific background can be reduced by the fact that the detection antibody does not bind directly to the bound Aß peptide, but to a secondary antibody, which recognizes the bound Analtyten and binds but is not enzyme-conjugated.
  • Alzheimer's disease in 2001 they observed that both in body fluids of healthy individuals and in body fluids of diseased Alzheimer's patients natural antibodies against A ⁇ peptides are present (compare Du et al., "Reduced levels of amyloid ß-peptide antibody in Alzheimer's disease", Neurology 2001), so-called amyloid beta autoantibodies (Aß autoantibodies) so-called autoantibodies, if Alzheimer's disease is present.
  • Aß autoantibodies amyloid beta autoantibodies
  • the object of the invention is therefore to provide an improved ELISA for the detection of Aß- Autoantiköpern available that allows rapid evaluation with simple and inexpensive means.
  • the ELISA should be quick and easy to perform, especially with human serum and / or plasma samples as well as CSF samples.
  • ELISA enzyme-linked immunosorbent assay
  • providing the antigen-coated solid phase comprises incubating the solid phase with a coating solution in which an antigen having a peptide sequence selected from the group SEQ ID NO. 1, SEQ ID no. 2 or SEQ ID No 3.
  • SEQ ID no. 2 or SEQ ID NO. 3 has, is coated. Surprisingly, such 40 to 46 amino acid-long peptides show a very fast
  • the autoantibodies to be detected in the ELISA therefore bind rapidly and reliably to the antigens immobilized on the solid phase. It is particularly advantageous if the antigen has a peptide sequence corresponding to SEQ ID no. 1 or SEQ ID NO. 2 has.
  • a particularly critical point in the establishment of an ELISA is, as stated above, the coating of the solid phase with the binding partner of the analyte.
  • the coating solution is a carbonate buffer with basic pH. It has thus been shown that the antigens according to the invention bind significantly better to the solid phase in a basic medium, in particular at a pH of 9.6, than in the conventionally used neutral medium at about pH 7.
  • the incubation of the solid phase with the coating solution can be carried out both at 4 0 C overnight, which is particularly advantageous when using an antigen having a sequence corresponding to SEQ ID NO. 1, as well as at 37 ° C and 5% CO 2 for 2 hours, which is preferred when using an antigen having a sequence corresponding to SEQ ID NO. 2 is the case.
  • blocking serves to cover the areas of the solid phase surface to which no antigen is bound, so that the analyte to be detected in the subsequent incubation with the sample can bind exclusively to the antigens and not to the solid phase itself.
  • the blocking solution is a Tris-buffered protein solution with pH 7.4, containing at least one anti-microbial agent.
  • the commercially available blocking solution Superblock TBS® or Superblock® from Pierce, Germany can be used.
  • the incubation with the blocking solution is preferably carried out at 4 ° C overnight. In this way, the free surface of the solid phase can be covered at the sites where no antigen is bound particularly well with the protein contained in the blocking solution. This effectively prevents antigens or antibodies present in the sample from nonspecifically binding to the solid phase, thus providing unwanted false-positive signals, also referred to as non-specific interfering background.
  • the solid phase is a microtiter plate.
  • This usually has several sections for the analysis of patient samples or standard and control, so-called wells. All wells of a microtiter plate can be prepared identically. Usually, all wells are simultaneously coated with antigen and then blocked. Subsequently, each particular wells are filled with a defined amount of standard, patient sample and control and treated further. The same volume of liquid is filled into each well. However, the respective concentration of standard, patient sample and control is varied by suitable dilution from well to well, in order to allow the most precise possible concentration determination of the analyte to be detected.
  • Another advantage of the invention is that the wells are filled with a relatively high total volume, namely up to 300 .mu.l, but which contains only a relatively small proportion of sample (ie either standard, patient sample or control).
  • an embodiment of the detection method according to the invention provides that the wells are first filled with 250 ⁇ l of an assay buffer. Subsequently, only 10 ⁇ l of the sample to be examined are added to this buffer.
  • the assay buffer serves for the defined dilution of the sample.
  • sample dilution buffer is preferably such that it does not interfere with the serum matrix upon dilution.
  • the wells are filled with 200 ⁇ l of prediluted sample. After filling the wells with the samples (ie standard, patient sample and control), the microtiter plate is incubated for 60 minutes on a shaker at 300 to 500 rpm and room temperature.
  • the incubation of the microtiter plate with the solution to be examined advantageously comprises the following steps: introduction of diluted sample into each well of the microtiter plate, wherein the introduction of the diluted sample is carried out so that thereafter in each well a total volume of 200 to 300 .mu.l Assay buffer containing the sample, and incubate for 60 minutes on a shaker at 300 to 500 rpm and room temperature.
  • the assay buffer is preferably a sodium phosphate buffer at pH 7.0, comprising 3 to 9% BSA, 0.01 to 3% TWEEN and at least one preservative selected from the group 5-bromo-5-nitro- 1,3-dioxane (BND), 2-chloroacetamide (CAA), 2-hydroxypyridine N-oxide (HPO), N-methylisothiazolone (MIT), sodium azide, thimerosal, proclin.
  • BND 5-bromo-5-nitro- 1,3-dioxane
  • CAA 2-chloroacetamide
  • HPO 2-hydroxypyridine N-oxide
  • MIT N-methylisothiazolone
  • the next step of the method of the invention is the immunological detection of the A ⁇ autoantibody captured by the binding partner on the solid phase. This is done by the following steps: tapping the contents out of the wells, washing the wells three to five times with in each case 300 to 500 .mu.l wash solution per well, removing remaining liquid drops by stripping the wells on a suction paper, introducing from 50 .mu.l to 100 .mu.l enzyme Conjugate in each well, incubate on a shaker at 300 to 500 rpm and room temperature for 30 to 60 minutes, Shake out the contents from the wells, wash the wells three to five times with each 300 to 500 .mu.l wash solution per well, remove remaining drops of liquid by wiping the wells on a suction paper, add 50 .mu.l substrate solution to each well, incubate for 15 to 20 minutes Room temperature and stopping the enzymatic reaction by adding 100 ⁇ l stop solution to each well.
  • the washing solution is preferably a Tris buffer containing 0.01 to 3% Tween.
  • a particular advantage of the ELISA according to the invention is that the binding kinetics of the A ⁇ autoantibody to the antigens according to the invention is so optimal that a chromogenic enzyme-substrate reaction can be used for immunological detection, the result of which is determined by determining the optical density
  • the enzyme conjugate introduced into the wells after the first wash contains the detection antibody. It is preferably an antibody directed against human IgG ( ⁇ -human IgG). This may, for example, come from one of the following species: goat, mouse, guinea pig, rat, donkey, cattle, sheep or pig. While others are generally less common, they are conceivable.
  • the a-human IgG antibody is conjugated with an enzyme or a dye so that it can be visualized either directly - if it is, for example, a fluorescent dye - or by reacting a substrate with the aid of the enzyme.
  • the enzyme may be, for example, an enzyme selected from the group consisting of peroxidase (POD), horseradish peroxidase (HRP), alkaline phosphatase (AP), ⁇ -galactosidase ( ⁇ -gal).
  • POD peroxidase
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • ⁇ -galactosidase ⁇ -gal
  • the enzyme conjugate contains an enzyme conjugated anti-human IgG antibody derived from a species selected from goat, mouse, guinea pig, rat, donkey, bovine, sheep, with the antibody conjugated to the antibody selected horseradish peroxidase (HRP), alkaline phosphatase (AP), ß-galactosidase (ß-Gal).
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • ß-Gal ß-galactosidase
  • Suitable substrates may for example be selected from the following group: 3,3'-diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC), 4-chloro-1-naphthol (CN), tetramethylbenzidine (TMB), vomuchsin, Naphthol AS-MX phosphate, 5-bromo-5-chloro-3-indoxyl phosphate (BCIP), nitroblue tetrazolium chloride (NBT), 5-bromo-4-chloro-3-indoxyl- ⁇ -D-galactopyranoside (X- GaI), 5-bromo-3-indolyl- ⁇ -D-galactopyranoside (Blue-Gal), 6-chloro-3-indolyl- ⁇ -D-galactopyranoside (Y-GaI), 5-iodo-3-indolyl- ⁇ -D-galactopyranoside (Purple-Gal),
  • the substrate solution contains at least one chromophore selected from the group 3,3'-diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC), 4-chloro-1-naphthol (CN), 3,3 ' , 5,5'-tetramethylbenzidine (TMB), damuchsin, naphthol AS-MX phosphate, 5-bromo-5-chloro-3-indoxyl phosphate (BCIP), nitroblue tetrazolium chloride (NBT), 5-bromo-4-chloro 3-indoxyl- ⁇ -D-galactopyranoside (X-GaI), 5-bromo-3-indolyl- ⁇ -D-galactopyranoside (Blue-Gal), ⁇ -chloro-S-indolyl- ⁇ -D-galactopyranoside (Y -GaI), 5-iodo-3-indolyl- ⁇
  • the substrate to be cleaved by the enzyme is usually added in excess, it is necessary to end the enzyme-substrate reaction in a controlled manner after a defined period of time. This can be done, for example, by adding stop solution. This changes the reaction conditions such that the enzyme-substrate reaction can not continue.
  • the reaction is preferably stopped by adding an acid, usually sulfuric acid.
  • the stop solution is then the simplest Case from the diluted acid. It can be seen that the stop solution has a pH of less than 7.0 and is preferably a dilute hydrochloric or sulfuric acid.
  • the invention also provides a microtiter plate having at least one well, each well containing a peptide corresponding to one of the sequences SEQ ID NO. 1 to 3 is coated.
  • a microtiter plate having at least one well, each well containing a peptide corresponding to one of the sequences SEQ ID NO. 1 to 3 is coated.
  • Such a microtiter plate can be used with great advantage in order to carry out the method according to the invention.
  • the microtiter plate has at least one test unit comprising 24 wells.
  • the 24 wells can then be divided into three groups.
  • the first eight wells are loaded with eight different concentrations of the standard. From the measured values obtained therefrom, the
  • Comparison curve can be determined. The second eight wells come with eight different ones
  • the microtiter plate has a plurality of test units which can be used independently of one another.
  • a large microtiter plate with a plurality of linearly successively arranged test units is conceivable, which can be bent or broken off with the aid of a perforation or groove before use of the microtiter plate.
  • microtiter plates whose wells have a flat bottom as well as wells with a round, v-shaped or c-shaped bottom are suitable for carrying out the method according to the invention. Men therefore recognized that it is also advantageous in the case of the microtiter plate according to the invention that the wells have a round, flat, V-shaped or C-shaped bottom.
  • a microtiter plate of the invention is particularly advantageously produced by a method comprising the steps of: providing an uncoated microtiter plate, incubating the microtiter plate with a coating solution consisting of a basic pH 9.6 carbonate buffer containing 5 ⁇ g / ml of a Antigen according to one of the sequences according to SEQ ID no. 1, 2 or 3, for two hours at 37 °, and incubating the microtiter plate with a blocking solution containing a pH 7.4 Tris-buffered protein solution at least one anti-microbial agent, at 4 ° C overnight.
  • microtiter plate according to the invention for use in a method according to the invention.
  • the invention further provides a test kit for detecting A ⁇ autoantibodies in serum samples, comprising at least one antigen-coated microtiter plate, wherein the antigen is a peptide whose sequence is a sequence selected from SEQ. ID. 1, 2 or 3 corresponds. It is particularly favorable if the test kit also contains ready-made reagents for carrying out the test. The operator then only has to add the corresponding serum sample to be examined and a corresponding appropriate control sample. It is also conceivable that the test kit already contains a selection of control samples. It will be appreciated that the test kit preferably contains a standard, assay buffer, wash solution, enzyme conjugate, substrate solution and / or stop solution.
  • test kit according to the invention is suitable for use in a method according to the invention.
  • FIG. 3 with the aid of the ELISA according to the invention, embodiment 3 corresponds to certain amyloid beta-autoantibody concentrations in two of the morbus
  • all three peptides which can be used according to the invention for coating the solid phase have at least the amino acids 1 to 40 of the naturally occurring A ⁇ peptide.
  • the framed sequence regions correspond in each case to the sequence of the A ⁇ 1-4 o-peptide, the gray-shaded sequence regions of the sequence of the A ⁇ 1-42 peptide.
  • Cys amyloid beta 1-42 and SEQ ID NO. 1 corresponding sequence also includes C-terminal amino acids 41 and 42 of the amyloid beta peptide (Aß) and N-terminal four additional amino acids, namely the tetrapeptide CGKR. It is therefore the case of SEQ ID no. 1 corresponding antigen to a total of 46 amino acid polypeptide, which is composed of the tetrapeptide CGKR followed by the amino acids 1 to 42 of
  • SEQ ID NO. 1 denotes a modified human peptide whose amino acids 5 to 46 represent the sequence of the naturally occurring human amyloid beta 1-42 peptide, to which is attached N-terminally the tetrapeptide Cys Gly Lys Arg containing amino acids 1 to 4 of SEQ ID No. 1 corresponds. It is further evident in FIG. 1 that SEQ ID no. 2, which is referred to as amyloid beta 1-42, the natural sequence of the amyloid beta 1-42 peptide equivalent. It is therefore the case of SEQ ID no. 2 corresponding antigen to a total of 42 amino acids existing polypeptide, which corresponds to the amino acids 1 to 42 of the Aß 1 ⁇ 2 peptide.
  • SEQ ID NO. Designated Cys amyloid beta 1-40.
  • Figure 3 corresponds to the natural amyloid beta 1-40 peptide (A ⁇ ⁇ o). However, the peptide at the N-terminus was modified by the attachment of a cysteine. It is therefore the case of SEQ ID no. 3 corresponding antigen to a total of 41 amino acid polypeptide, which is composed of the amino acids 1 to 40 of the Aß ⁇ o-peptide and an N-terminal added cysteine.
  • SEQ ID NO. Designated Cys amyloid beta 1-40.
  • Figure 3 corresponds to the natural amyloid beta 1-40 peptide (A ⁇ ⁇ o). However, the peptide at the N-terminus was modified by the attachment of a cysteine. It is therefore the case of SEQ ID no. 3 corresponding antigen to a total of 41 amino acid polypeptide, which is composed of the amino acids 1 to 40 of the Aß ⁇ o-peptide and an N-terminal added cysteine.
  • FIG. 2 shows the OD 450 measured values of a method according to the invention carried out in accordance with the protocol of exemplary embodiment 3.
  • FIG. 3 illustrates that data obtained with the aid of the method according to the invention can be used inter alia for the diagnosis of Alzheimer's disease or other neurodegenerative diseases.
  • microtiter plates with an antigen corresponding to SEQ ID no. 1, ie an Aß peptide comprising amino acids 1 to 42 and N-terminal additionally carries the amino acid sequence CGKR coated.
  • the antigen is dissolved in a carbonate buffer with pH 9.6. After incubation with the coating solution thus prepared, the microtiter plate is incubated with blocking solution.
  • sample which is either the standard, a control or the sample actually to be assayed, to the assay buffer.
  • sample is thus diluted in assay buffer such that, in the end, there is a volume of 260 ⁇ l of diluted sample in each well.
  • the plate with the thus diluted samples is incubated for 60 minutes on a shaker at 300 to 500 rpm at room temperature.
  • the contents of the wells are immediately poured out and washed each well 3 times with 400 .mu.l washing solution.
  • the wells are tapped on absorbent paper.
  • Embodiment 2 essentially corresponds to embodiment 1 already described above. However, the wells are loaded with a total volume of 200 ⁇ l of diluted sample. After removing the sample, the wells are each washed five times with wash solution, then 100 ⁇ l enzyme conjugate are added to each well.
  • the reaction of the enzyme with the chromophore occurs during a 15 minute incubation at room temperature.
  • an antigen corresponding to SEQ ID no. 2, so the peptide A ⁇ 1-42 diluted in a concentration of 5 ug / ml in 0.1 M sodium carbonate buffer of pH 9.6.
  • This solution is used to incubate a solid phase, namely a 96-well-high-binding ELISA plate, for 2 hours at 37 ° C. in a CO 2 incubator at 5% CO 2 . Subsequently, the plate is incubated overnight with a commercially available blocking solution. The thus prepared plate is washed four times with a washing solution according to the invention.
  • an HRP-coupled anti-human second antibody is used for detection of A ⁇ autoantibody bound on the plate. This is diluted 1: 4000 in the previously used blocking solution.
  • the chromogenic substrate used is TMB.
  • the color reaction is stopped with the aid of sulfuric acid and evaluated at a wavelength of 450 nm by determining the OD.
  • the OD of the standards is first determined and then compared with the samples or controls. For this purpose, a comparison curve is first determined using a defined concentration series of the standard. The samples and controls are applied several times and at different dilutions.
  • microtiter plate The exemplary loading of a microtiter plate can look like this:
  • Wells 1 to 8 Standard in different concentrations to create a
  • Wells 17 to 24 Sample of a potentially ill person to be measured at various dilutions
  • the design of the test may be varied as needed.
  • a total of 40 wells to be measured in a microtiter plate were loaded as shown in table 1.
  • the first 16 wells are loaded with a standard dilution series to create a comparison curve from which the OD readings of the samples can be used to calculate the concentration of A ⁇ autoantibodies present in the samples.
  • Table 1 Loading scheme for 40 wells of a microtiter plate according to the invention:
  • Wells 17 to 24 serial dilution of a first diseased person serum
  • wells 25 to 32 dilution series of a second diseased person serum sample
  • wells 33 to 40 serial dilution of a healthy control subject serum sample.
  • the wells of the microtiter plate loaded with sample or standard as shown in Table 1 are incubated for 4 hours at 37 ° C. according to the protocol described above, then washed, incubated with enzyme conjugate, washed again and incubated with substrate solution. After stopping the enzyme-substrate reaction, the OD 450 is determined with a corresponding reader.
  • FIG. 2 a shows the measured values of such an OD 450 determination.
  • Curve 1 shows the OD readings of the wells loaded with the standard set
  • curve 2 the measured values of the diluted serum samples of the diseased person 1
  • curve 3 the measured values of the diluted serum samples of the diseased person 2
  • curve 4 the measured values of the diluted serum Samples of the control person.
  • the concentrations of A ⁇ autoantibodies in the respective serum samples can be calculated from the OD 450 values following the measurement.
  • both diseased persons each have more than 900 ug / ml Aß autoantibodies in the serum.
  • the healthy control subject shows a significantly lower value of only about 840 ⁇ g / ml.
  • 96-well ELISA plates with an antigen corresponding to SEQ ID no. 3, ie an A ⁇ ⁇ peptide, which is N-terminally provided with a cysteine coated ie an A ⁇ ⁇ peptide, which is N-terminally provided with a cysteine coated.
  • the ELISA plates are incubated at 4 C C overnight with a coating solution.
  • a coating solution 5 ⁇ g / ml of antigen are dissolved in a 100 mM sodium bicarbonate buffer of pH 9.6 immediately before incubation.
  • the plates are incubated with blocking solution for 2 hours at room temperature, or alternatively at 4 C C overnight.
  • the blocking solution used is a commercially available solution, for example SuperBlock from Pierce, Germany.
  • human IVIgG affinity purified autoantibodies are diluted with saline, which has a pH of 7.4, in concentrations from 50 to 0.78 ⁇ g / ml.
  • the standards thus prepared are incubated at 4 ° C overnight on the antigen-coated plates.
  • the second antibody used is a goat HRP-conjugated ⁇ -human antibody which specifically recognizes the heavy and light chains of immunoglobulins (HRP-labeled H & L chain specific Goat anti-human from Calbiochem, Darmstadt, Germany).
  • the chromogenic substrate used is 3,3 ', 5,5'-tetramethylbenzidine (TMB).
  • TMB 3,3 ', 5,5'-tetramethylbenzidine
  • the enzymatic reaction is terminated with 2N sulfuric acid (H 2 SO 4 ).
  • the plates are evaluated at 450 nm using a Thermo Electron Corp reader.
  • the invention comprises a test kit for carrying out an ELISA according to the invention.
  • the test kit contains a microtiter plate coated with a peptide having a sequence corresponding to SED ID no. 1 has and as
  • Antigen is the immobilized binding partner for the Aß autoantibodies to be detected.
  • the kit also contains blocking solution, wash solution, assay buffer, concentrated amount of standard, enzyme conjugate, substrate and stop solution.
  • the invention is not limited to one of the above-described embodiments, but can be modified in many ways. It will be appreciated that in a method, in particular enzyme-linked immunosorbent assay (ELISA), for the in vitro detection of A ⁇ autoantibodies in human serum and / or plasma, comprising the steps of providing an antigen-coated solid phase, incubating the solid phase with a Blocking solution, incubating the solid phase with a sample to be examined, immunological detection of Aß- autoantibody on the solid phase and reading the detection result on the solid phase using a reader is advantageous if the provision of the antigen coated solid phase comprises incubating the solid phase with a coating solution in which an antigen is dissolved, which contains a peptide sequence selected from the group SEQ ID NO.
  • ELISA enzyme-linked immunosorbent assay
  • the antigen is preferably a peptide sequence corresponding to SEQ ID no. 1 has.
  • the coating solution is a basic pH carbonate buffer and if the blocking solution is a pH 7.4 Tris-buffered protein solution containing at least one anti-microbial agent. Incubation of the solid phase with the coating solution is carried out at 4 ° C overnight or at 37 ° C and 5% CO 2 for 2 hours. Incubation with the blocking solution is carried out at 4 ° C overnight.
  • the solid phase is preferably a microtiter plate and the incubation of the microtiter plate with the solution to be examined comprises the following steps: introduction of diluted sample into each well of the microtiter plate, wherein the introduction of the diluted sample is carried out such that thereafter in each well a total volume of 200 to 300 ⁇ l of assay buffer containing the sample, incubate for 60 minutes on a shaker at 300 to 500 rpm and room temperature.
  • the immunological detection of the A ⁇ autoantibody comprises the following steps: tapping the contents out of the wells, washing the wells three to five times with each 300 to 500 ⁇ l washing solution per well, removing remaining liquid drops by stripping the dishes on a suction paper, introducing 50 ⁇ l to 100 ⁇ l of enzyme
  • the reading of the detection result is carried out at 450 ⁇ 10 nm, preferably within 10 minutes after the addition of the stop solution.
  • the assay buffer is a sodium phosphate buffer at pH 7.0, comprising 3 to 9% BSA, 0.01 to 3% TWEEN and at least one
  • Preservatives selected from the group consisting of 5-bromo-5-nitro-1,3-dioxane
  • each well is reacted with a peptide corresponding to one of the sequences SEQ ID NO. 1 to 3 is coated.
  • the peptide corresponding to one of the sequences SEQ ID NO. 1 to 3 is coated.
  • Microtiter plate at least one test unit comprising 24 wells.
  • the test unit comprising 24 wells.
  • Microtiter plate very particularly preferably a plurality of test units, which are used independently of each other.
  • the wells may have a round, flat, V-shaped or C-shaped bottom.
  • a microtiter plate according to the invention is according to a
  • a process prepared comprising the steps of: providing an uncoated one
  • Microtiter plate incubate the microtiter plate with a coating solution consisting of a basic pH 9.6 carbonate buffer in which 5 ⁇ g / ml of an antigen corresponding to one of the sequences according to SEQ ID no. 1, 2 or 3 for two hours at 37 °, and incubate the microtiter plate with a blocking solution containing a pH 7.4 Tris-buffered protein solution containing at least one anti-microbial agent at 4 ° C overnight.
  • a coating solution consisting of a basic pH 9.6 carbonate buffer in which 5 ⁇ g / ml of an antigen corresponding to one of the sequences according to SEQ ID no. 1, 2 or 3 for two hours at 37 °
  • a blocking solution containing a pH 7.4 Tris-buffered protein solution containing at least one anti-microbial agent at 4 ° C overnight.
  • a test kit for detecting A ⁇ autoantibodies in serum samples at least comprising an antigen-coated microtiter plate, wherein the antigen is a peptide whose sequence is a sequence selected from SEQ. ID. 1, 2 or 3 corresponds to an advantageous embodiment of the invention. It is advantageous if the test kit contains assay buffer, wash solution, enzyme conjugate, substrate solution and / or stop solution.
  • the test kit according to the invention can advantageously be used in a method according to the invention.
  • amyloid beta-peptide comprising amino acids 1 to 40
  • a ⁇ 1-42 amyloid beta-peptide comprising amino acids 1 to 42
  • CSF Cerebrospinal Fluid

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Abstract

L'invention concerne un procédé, notamment un dosage par la méthode ELISA, pour détecter in vitro des auto-anticorps Aß dans du sérum et/ou plasma humain, comprenant les étapes de préparation d'une phase solide revêtue d'antigène, d'incubation de la phase solide avec une solution de blocage, d'incubation de la phase solide avec un échantillon à analyser, de la détection immunologique de l'auto-anticorps Aß sur la phase solide et de lecture des résultats de détection sur la phase solide à l'aide d'un appareil de lecture. Selon l'invention, il est avantageux que la préparation de la phase solide revêtue d'antigène comprenne l'incubation de la phase solide avec une solution de revêtement dans laquelle est dissous un antigène qui présente une séquence peptide sélectionnée dans le groupe consistant en SEQ ID NO. 1, SEQ ID NO. 2 ou SEQ ID NO. 3.
EP10747569A 2009-07-20 2010-07-14 Procédé, notamment un dosage par la méthode elisa, pour détecter in vitro des auto-anticorps bêta amyloïde, plaque de microtitration et kit d'essai Withdrawn EP2457093A1 (fr)

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DE102009034119A DE102009034119A1 (de) 2009-07-20 2009-07-20 Verfahren, insbesondere Enzyme-linked Immunosorbent Assay (ELISA), zum in vitro Nachweis von Amyloid beta Autoantikörpern, Mikrotiterplatte und Testkit
PCT/DE2010/000817 WO2011009435A1 (fr) 2009-07-20 2010-07-14 Procédé, notamment un dosage par la méthode elisa, pour détecter in vitro des auto-anticorps bêta amyloïde, plaque de microtitration et kit d'essai

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EP2457093A1 true EP2457093A1 (fr) 2012-05-30

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ITRM20120383A1 (it) 2012-03-20 2013-09-21 Uni Degli Studi Di Milano B Icocca Metodo e kit per la rivelazione di anticorpi.
CN109490539A (zh) * 2018-12-28 2019-03-19 广州动佰生物科技有限公司 检测某种动物病原抗体的elisa试剂盒
CN113281502B (zh) * 2021-05-14 2024-02-02 遵义医科大学附属医院 一种酶联免疫吸附试验模拟方法

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US20060188951A1 (en) * 2003-02-24 2006-08-24 In Hee Mook Method for measuring the level of anti-beta-amyloid antibody in body fluids and diagnostic kit for alzheimer's disease using same
DK2104682T3 (en) * 2007-01-11 2017-01-16 Michael Bacher DIAGNOSIS AND TREATMENT OF ALZHEIMER'S AND OTHER DEMENTIA DISEASES
US20110027288A1 (en) * 2008-03-26 2011-02-03 Moses Rodriguez Human monoclonal antibodies against amyloid beta protein, and their use as therapeutic agents
EP2143447A1 (fr) * 2008-07-11 2010-01-13 Universität Konstanz Bioconjugués comportant des épitopes spécifiques aux anticorps Aß pour une immunothérapie et diagnostic pour la maladie d'Alzheimer

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WO2004056318A2 (fr) * 2002-12-19 2004-07-08 New York University Methode permettant de traiter une maladie amyloide

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SOHN ET AL: "Reduced serum level of antibodies against amyloid beta peptide is associated with aging in Tg2576 mice", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 361, no. 3, 11 August 2007 (2007-08-11), pages 800 - 804, XP022196499, ISSN: 0006-291X, DOI: 10.1016/J.BBRC.2007.07.107 *

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