EP1388012A2 - Quantitaver einschritt immuntest in lyophilisierter form - Google Patents

Quantitaver einschritt immuntest in lyophilisierter form

Info

Publication number
EP1388012A2
EP1388012A2 EP02769103A EP02769103A EP1388012A2 EP 1388012 A2 EP1388012 A2 EP 1388012A2 EP 02769103 A EP02769103 A EP 02769103A EP 02769103 A EP02769103 A EP 02769103A EP 1388012 A2 EP1388012 A2 EP 1388012A2
Authority
EP
European Patent Office
Prior art keywords
detection
acid
microtiter plate
plate
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Withdrawn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
EP02769103A
Other languages
German (de)
English (en)
French (fr)
Inventor
Irene Rech-Weichselbraun
Michael Schaude
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.)
MEDSYSTEMS DIAGNOSTICS GmbH
Original Assignee
MEDSYSTEMS DIAGNOSTICS GmbH
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 MEDSYSTEMS DIAGNOSTICS GmbH filed Critical MEDSYSTEMS DIAGNOSTICS GmbH
Publication of EP1388012A2 publication Critical patent/EP1388012A2/de
Withdrawn legal-status Critical Current

Links

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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • 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
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent

Definitions

  • the invention relates to an immunoassay kit for the qualitative and quantitative determination of a sample by -specifically-binding binders such as - - ⁇ - B - - - Anti-kerper- 7 - -A t -ige-ne -, - receptors and ligands, with a carrier plate or a microtiter plate with a plurality of wells, the carrier plate or the microtiter plate being precoated with a primary binding partner and the binding partner being fixed as a lyophilisate.
  • binders such as - - ⁇ - B - - - Anti-kerper- 7 - -A t -ige-ne -, - receptors and ligands
  • Immunoassays enzyme-linked immunoassays, fluorescence-label-linked immunoassays, luminescence-label-linked immunoassays or immunoassays labeled with another label are used in routine diagnostics and research diagnostics for the detection and quantification of proteins, e.g. antigens, ligands, receptors, antibodies and chemical compounds .
  • the state of the art in such test kits is to bind one of the binding partners to a solid support, for example a polystyrene plate, and the other reagents required for carrying out the test as separate. Add components of the test kit.
  • reagents are usually the protein to be detected in quantified form as concentrate or lyophilisate (reference standard), a standard and sample dilution medium, the second specific binding partner in coupled form (conjugate) as concentrate or working solution, if required a secondary detection system and in the case of an enzyme label this substrate reagent required for detection.
  • a stop solution is added to terminate the enzymatic reaction, and an assay buffer, which is usually present as a concentrate, is available to prepare the working solutions for the second specific binding partner and for the secondary reagent. Since washing steps are required between the work steps, a conventional immunoassay kit contains a washing buffer, usually in a concentrated form.
  • Commercial ELISA kits offer the user validated test systems.
  • the products contain all the reagents required for carrying out the test in optimized concentrations and amounts as well as a detailed protocol for carrying out • the - test -. the - usual-white e-consecutive carried out.
  • the common incubation of analytes (standard, sample) and secondary antibody with the solid phase (cocktail) is possible for a large number of test systems.
  • a typical example of an immunoassay is the sandwich ELISA (enzyme linked immunosorbent assay).
  • Such ELISA kits typically contain the following components:
  • Reference material analyte (standard) in a defined concentration to create a standard series
  • Assay buffer saline solution for diluting the secondary antibody and streptavidin enzyme concentrate (preparation of working solutions)
  • Sample dilution medium Specific liquid medium for dilution of the reference material and the unknown samples
  • the invention now aims to simplify the handling of such test kits and, in addition to a qualitative statement, also to enable quantitative statements to be made.
  • the invention also aims to reduce the likelihood of errors for the user by minimizing the work steps to be carried out.
  • the aim of the present invention is to reduce the packaging volumes and packaging costs as well as the shipping costs by significantly reducing the weight and volume of the kits and to provide a logistical simplification in which standardized basic kits are provided with improved shelf life of the product.
  • the immunoassay kit according to the invention essentially consists in that a reference standard series of the sample to be determined with incremental dilution in lyophilized form is additionally present in part of the wells of the microtiter plate.
  • a reference standard series of the sample to be determined with incremental dilution in lyophilized form is additionally present in part of the wells of the microtiter plate.
  • the prefabrication of the immunoassay kit can be pushed much further and the necessary determination steps for the sample can be further reduced.
  • the design is advantageously made such that the wells of the microtiter plate additionally contain an enzyme or biotin-coupled conjugate of a secondary binding partner, in particular an antibody, and in the case of the biotin-coupled conjugate, an enzyme in lyophilized form.
  • the second specific binding partner conjugate in titrated concentration and, if necessary, the secondary reagent coupled with one of the above-mentioned labels is also used for detection together with the primary binding partner which is firmly bound to phases already introduced in lyophilized form in the solid phase, the provision of these components in lyophilized form, i. H . thus in a form dehydrated at low temperatures of about -30 ° C., the reaction kinetics freeze to such an extent that there is no fear of a preliminary reaction of the reference material. Because highly diluted working solutions just as limited stable solutions are not kept in stock, but rather are generated on the carrier immediately after the start of the test by adding solvent or rehydration, further possible sources of error are excluded.
  • the design is such that the wells of the microtiter plate contain a sample dilution medium in lyophilized form.
  • the specific sample dilution medium can be introduced into the corresponding wells of the microtiter plate at a temperature of, for example, 2 ° C., the secondary antibody-enzyme conjugate or the mixture of secondary antibody-biotin conjugate being able to be introduced into all wells of the microtiter plate at the same temperature , whereupon the microtiter plate charged in this way is subjected to a lyophilization in a freeze-drying process in a freeze-dryer, for example, which has been pre-cooled to -30 ° C.
  • Such a prefabricated carrier or such a prefabricated microtiter plate only requires a small number of additional components to complete the test kit, the test kit advantageously containing only washing buffer, substrate solution and stop solution in addition to the precoated microtiter plate.
  • the test kit advantageously containing only washing buffer, substrate solution and stop solution in addition to the precoated microtiter plate.
  • For the determination of the sample it is therefore only necessary to rehydrate the microtiter plate by adding defined volumes of distilled water to the wells of the standard series, if necessary the blanks and the sample wells, whereupon the unknown sample is added and incubated.
  • the stop solution is added after a predetermined time and the test evaluation can be carried out immediately.
  • the training is advantageously made such that streptavidin - horse radical peroxidase (HRP) is used as the secondary reagent in the case of biotin-coupled secondary antibody conjugate.
  • HRP horse radical peroxidase
  • test execution for the user is reduced to the addition of the unknown sample and the enzymatic reaction by the inventive design, all further steps being carried out beforehand by the manufacturer of the ELISA kit.
  • the microtiter plate is thus coated, blocked and fixed at the manufacturer with the specific primary antibody, whereupon the plate is cooled, for example, to -20 ° C. and the additional steps described above for introducing the standard series of the sample diluent and the secondary antibody-enzyme conjugate or the mixture made of secondary antibody-biotin conjugates and streptavidin enzyme in a defined manner at the manufacturer.
  • the test procedure is therefore reduced to the addition of the samples to be analyzed and the test evaluation based on the substrate staining.
  • the immunoassay kit according to the present invention is designed such that the test kit stabilizers for peroxidase or general lyoprotectants such as proteins (e.g. albumin (e.g. BSA) protein hydrolysates (peptones, gelatin ...) casein), Polymers (e.g. dextran, PVA, PVP), sugar (e.g. sucrose, trehalose, lactose, xylitol, sorbitol, mannitol, maltose, glucose, inositol), bacteriostatic agents (e.g.
  • proteins e.g. albumin (e.g. BSA) protein hydrolysates (peptones, gelatin ...) casein
  • Polymers e.g. dextran, PVA, PVP
  • sugar e.g. sucrose, trehalose, lactose, xylitol, sorbitol, mannitol, maltose, glucose, inosito
  • phenolic substances and anilines with substituents small Alkyl radicals or Cl, Br ...) (e.g. o-methoxyphenol, o-methylphenol, p-methylphenol, o-aminophenol, o-hydroxybenzoic acid, (o, m or p) -hydroxybenzyl alcohol, aniline, p- Aminobenzoic acid, p-methoxyaniline, benzyl alcohol, benzoic acid, p-nitrophenol, benzylamine, 1-phenyl-l, 2-ethanediol, trans-1, 2-cyclohexanediol, cis-1, 2-cyclohexanedicarboxylic acid, cyclohexylamine); Hydrophobic compounds and Compounds and solvents (eg DMF, ethylene glycol, DMSO); Detergents (e.g. Tween-20); Aryl boric acid compounds (e.g. phenyl boric acid
  • TMB Luminol
  • Poly-hydroxy compounds e.g. polyols, polyethylene glycol, glycerin
  • Ectoins e.g. (S) -2-methyl-1,4,5,6-tetrahydropyrimidine-4-carbo-ylicacid [HP (
  • metal ions Al, Zn, Mg, Fe, Cu, ...)
  • Complexing agents e.g. EDTA
  • Amino acids e.g. glycine, prplin, 4-hydroxyproline, serine, glutamate, alanine, lysine, sarcosine, y-aminobutyruc acid, phenylalani
  • TRIS salts, aines, Na cholates, sucrose monolaurate, 2-O- contains ⁇ -mannosylglycerate.
  • the present immunoassay kit is suitable for a large number of possible applications.
  • Such an immunoassay kit is used in a particularly advantageous manner for use in research diagnostics and in-vitro diagnostics, for use in pathogen serology, such as, for example, for the detection of infections by HIV, HepV, EBV, CMV etc. for use in tumor diagnostics, for example for the detection of tumor markers or tumor-associated proteins, vascular risk markers, metastatic markers etc., for use in allergology such as for example for the detection of animal allergens, plant pollen, food components etc.
  • pathogen serology such as, for example, for the detection of infections by HIV, HepV, EBV, CMV etc.
  • tumor diagnostics for example for the detection of tumor markers or tumor-associated proteins, vascular risk markers, metastatic markers etc.
  • allergology such as for example for the detection of animal allergens, plant pollen, food components etc.
  • autoimmune diagnostics such as for the detection of autoantigens in the area of ANA / ENA, diabetes mellitus / IDDM, thyroid antigens, autoimmune hepatitis, APS, etc.
  • immunological disorders caused by inflammatory processes or infections such as Example for the detection of cytokines, adhesion molecules, chemokines, etc., for the detection of changes of the hormone balance such as ovulation tests, pregnancy tests, etc.
  • therapy monitoring such as for the detection of therapeutic antibodies and antigens, organic and inorganic compounds, for the detection of cell death by apoptosis or necrosis and / or for the detection of genetic changes, genetic diseases.
  • the invention can be used for the detection of antigens using a pair of antibodies (sandwich ELISA).
  • the detection of antibodies with the corresponding antigen is also possible.
  • the detection of receptors or ligands by the respective binding partner is a further possibility of using the present invention, so that a large number of test formats are available.
  • the test can be used to detect proteins, steroids, chemical compounds, drugs, nucleic acids, and similar substances.
  • the reaction complex can be detected with the aid of a directly coupled enzyme (for example horse radish peroxidase, alkaline phosphatase, etc.), via an enzymatic reaction which is carried out by a secondary step (biotin-streptavidin-HRP, enzyme-coupled antibody against the detec- tion antibodies and others).
  • a directly coupled enzyme for example horse radish peroxidase, alkaline phosphatase, etc.
  • biotin-streptavidin-HRP enzyme-coupled antibody against the detec- tion antibodies and others.
  • Each additional label that is suitable for carrying out an immunoassay such as fluorochromes, chemiluminescence labels, radioactive labels, and the like. a. , can also be used.
  • the carrier for immobilizing the primary binding partner is preferably a polystyrene 96-well microtiter plate, but any other carrier which is suitable for carrying out an immunoassay can also be used for the present invention.
  • the samples used for the measurement can include all liquid samples that contain the analyte, in particular body fluids such as sera, plasma preparations, local body liquids, whole blood, etc. and cell culture supernatants, buffered solutions of the analytes, among others.
  • body fluids such as sera, plasma preparations, local body liquids, whole blood, etc.
  • cell culture supernatants such as cell culture supernatants, buffered solutions of the analytes, among others.
  • the coated plate is used in the frozen state.
  • the sample dilution medium is cooled to 2 ° C.
  • 100 ⁇ l sample dilution medium is placed in each well in the first two rows of the microtiter plate submitted .
  • 100 ⁇ l sample dilution medium is placed in the corresponding wells.
  • 90 ⁇ l sample dilution medium is placed in each corresponding well.
  • the working solution (20ng / ml) of the reference material (2 ° C) is introduced in duplicate into the two uppermost wells on the left side of the microtiter plate (100 ⁇ l / well).
  • a standard series is prepared by serial 1: 2 dilution in the plate (10ng / ml; 5ng / ml; 2, 5ng / ml; 1, 25ng / ml; 0, 63ng / ml).
  • the microtiter plate is covered with all components with a film and placed in the freeze-dryer, which has been pre-cooled to -30 ° C. Lyophilization takes place for approx. 20 hours at -30 ° C.
  • the dried plate is welded into an aluminum bag together with the desiccant immediately after removal from the freeze dryer.
  • the plate is taken out of the aluminum bag.
  • the standard series and the blanks are rehydrated by adding 150 ⁇ l distilled water per well, the sample wells by adding 140 ⁇ l H20. 10 ⁇ l of the unknown sample are applied to the sample wells.
  • the plate is covered with a film and incubated for 1 hour at RT. The plate is washed 3 times, 100 ⁇ l of substrate solution is added to each well of the plate.
  • the enzyme reaction is after Interrupted for 15 minutes by adding the stop solution (100 ⁇ l) and the color intensity in the individual wells was evaluated photometrically.
  • Step 1 review:
  • the coated plate is used in the frozen state.
  • the sample dilution medium is cooled to 2 ° C.
  • the first two rows of the microtiter plate are diluted with 100 ⁇ l Pr in each well - medium presented.
  • 100 ⁇ l sample of dilution medium is placed in the corresponding wells.
  • 50 ⁇ l sample dilution medium is placed in each well.
  • the working solution (400pg / ml) of the reference material (recombinant human IL-10) (2 ° C) is introduced in duplicate into the two uppermost wells on the left side of the microtiter plate (100 ⁇ l / well).
  • a standard series (200-3.1 pg / ml) is prepared by serial 1: 2 dilution in the plate.
  • the microtiter plate is covered with all components with a film and placed in the freeze-dryer, which has been pre-cooled to -30 ° C. Lyophilization takes place for approx. 20 hours at -30 ° C. The dried plate is welded into an aluminum pocket together with the desiccant immediately after removal from the freeze dryer.
  • the plate is taken out of the aluminum bag.
  • the standard series and the blanks are rehydrated by adding 150 ⁇ l distilled water per well, the sample wells by adding lOO ⁇ l H20. 50 ⁇ l of the unknown sample are applied to each of the sample wells.
  • the plate is covered with a film and incubated at RT for 3 hours. The plate is washed 3 times, 100 ⁇ l of substrate solution is added to each well of the plate. The enzyme reaction is stopped after 15 minutes by adding the stop solution (l O O ⁇ l) canceled and the color intensity in the individual wells was evaluated photometrically.
  • Step 1
  • Specific coating / blocking The streptavidin coating solution is suctioned off, the plate is washed once with washing buffer (PBS / Tween). The plate is coated or blocked with 300 ⁇ l per well IFN ⁇ -biotin conjugate, l ⁇ g / l in PBS / 2% BSA for specific coating of the plate and for saturation of the polystyrene surface (prevention of non-specific binding) (2h, 37 ° C).
  • Step 3
  • the coated plate is used in the frozen state.
  • the sample dilution medium is cooled to 2 ° C. To create the standard series, the
  • Plate is made a standard series (100 - 1, 6ng / ml).
  • the microtiter plate is covered with all components with a film and placed in the freeze-dryer, which has been pre-cooled to -30 ° C. Lyophilization takes place for approx. 20 hours at -30 ° C. The dried plate is welded into an aluminum pocket together with the desiccant immediately after removal from the freeze dryer.
  • the plate is taken out of the aluminum bag.
  • the standard series and the blanks are rehydrated by adding 150 ⁇ l distilled water per well, the sample wells by adding 125 ⁇ l H20. 25 ⁇ l of the unknown sample are applied to the sample wells.
  • the plate is covered with a film and incubated for 2 hours at RT. The plate is washed 3 times, 100 ⁇ l of substrate solution is added to each well of the plate. The enzyme reaction is stopped after 15 minutes by adding the stop solution (100 ⁇ l) and the color intensity in the individual wells is evaluated photometrically.
  • Example of use 4 Example of use 4
  • BioLISA for the detection of human tumor necrosis factor alpha (TNF ⁇ ) (receptor-ligand binding)
  • the coated plate is used in the frozen state.
  • the sample dilution medium is cooled to 2 ° C.
  • 100 ⁇ l sample dilution medium is placed in each well in the first two rows of the microtiter plate.
  • 10 O ⁇ l sample dilution medium is placed in the appropriate wells.
  • 50 ⁇ l sample dilution medium is placed in each well.
  • the microtiter plate is covered with all components with a film and placed in the freeze-dryer, which has been pre-cooled to -30 ° C. Lyophilization takes place for approx. 20 hours at -30 ° C.
  • the dried plate is welded together with the desiccant into aluminum pockets immediately after removal from the freeze dryer.
  • the plate is taken out of the aluminum bag.
  • the standard series and the blanks are rehydrated by adding 150 ⁇ l distilled water per well, the sample wells by adding lOO ⁇ l H20. 50 ⁇ l of the unknown sample are applied to the sample wells.
  • the plate is covered with a film and incubated overnight at 4 ° C. The plate is washed 3 times, 100 ⁇ l of substrate solution is added to each well of the plate. The enzyme reaction is stopped after 15 minutes by adding the stop solution (100 ⁇ l) and the color intensity in the individual wells is evaluated photometrically.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)
EP02769103A 2001-05-10 2002-04-26 Quantitaver einschritt immuntest in lyophilisierter form Withdrawn EP1388012A2 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
AT3702001U 2001-05-10
AT0037001U AT5044U1 (de) 2001-05-10 2001-05-10 Quantitativer einschritt immuntest in lyophilisierter form
PCT/AT2002/000112 WO2002090983A2 (de) 2001-05-10 2002-04-26 Quantitaver einschritt immuntest in lyophilisierter form

Publications (1)

Publication Number Publication Date
EP1388012A2 true EP1388012A2 (de) 2004-02-11

Family

ID=3488777

Family Applications (1)

Application Number Title Priority Date Filing Date
EP02769103A Withdrawn EP1388012A2 (de) 2001-05-10 2002-04-26 Quantitaver einschritt immuntest in lyophilisierter form

Country Status (12)

Country Link
US (1) US20040171087A1 (sk)
EP (1) EP1388012A2 (sk)
JP (1) JP2004527758A (sk)
CN (1) CN1507565A (sk)
AT (1) AT5044U1 (sk)
CA (1) CA2446345A1 (sk)
CZ (1) CZ20033209A3 (sk)
HU (1) HUP0400081A3 (sk)
IL (1) IL158393A0 (sk)
PL (1) PL366665A1 (sk)
SK (1) SK13012003A3 (sk)
WO (1) WO2002090983A2 (sk)

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CA2446345A1 (en) 2002-11-14
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AT5044U1 (de) 2002-02-25
WO2002090983A3 (de) 2003-09-12
HUP0400081A3 (en) 2004-05-28
US20040171087A1 (en) 2004-09-02
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CZ20033209A3 (en) 2004-04-14
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