EP1388012A2 - Quantitative single-step immunoassay in lyophilised form - Google Patents
Quantitative single-step immunoassay in lyophilised formInfo
- 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- Withdrawn
Links
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- 102000043559 human ICAM1 Human genes 0.000 description 2
- 102000057041 human TNF Human genes 0.000 description 2
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- YWDAXANQVARVPC-UHFFFAOYSA-N (2-acetamidophenoxy)boronic acid Chemical compound CC(=O)NC1=CC=CC=C1OB(O)O YWDAXANQVARVPC-UHFFFAOYSA-N 0.000 description 1
- QBLFZIBJXUQVRF-UHFFFAOYSA-N (4-bromophenyl)boronic acid Chemical compound OB(O)C1=CC=C(Br)C=C1 QBLFZIBJXUQVRF-UHFFFAOYSA-N 0.000 description 1
- DDXCFDOPXBPUJC-SAYMMRJXSA-N 2-(alpha-D-mannosyl)-D-glyceric acid Chemical compound OC[C@H](C(O)=O)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]1O DDXCFDOPXBPUJC-SAYMMRJXSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 101001033233 Homo sapiens Interleukin-10 Proteins 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
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- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- KGUHOFWIXKIURA-VQXBOQCVSA-N [(2r,3s,4s,5r,6r)-6-[(2s,3s,4s,5r)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy-3,4,5-trihydroxyoxan-2-yl]methyl dodecanoate Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](COC(=O)CCCCCCCCCCC)O[C@@H]1O[C@@]1(CO)[C@@H](O)[C@H](O)[C@@H](CO)O1 KGUHOFWIXKIURA-VQXBOQCVSA-N 0.000 description 1
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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
- G01N33/543—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.
Abstract
An immunoassay kit for the qualitative and quantitative determination of a sample by means of specific binding partners, such as, for example, antibodies, antigens, receptors and ligands comprises a support or microtitre plate with a number of recesses. Said support or microtitre plate is pre-coated with a primary binding partner and the binding partner is applied and fixed as a lyophilisate. In addition a reference standard series of the sample to be determined lies in a part of the recesses of the microtitre plate with incremental dilution in lyophilised form.
Description
Quantitativer Einschritt Immuntest in lvophilisierter FormQuantitative one-step immunoassay in lvophilized form
Die Erfindung bezieht sich auf einen Immun-Test-Kit für die qualitative und quantitative Bestimmung einer Probe durch -spez-i-f-ische -Bindungspa-rfener wie-— z~B— - -An-t-i-kerper-7 — -A t-ige-ne-,— Rezeptoren und Liganden, mit einer Träger- bzw. einer Mikrotiterplatte mit einer Mehrzahl von Vertiefungen, wobei der Träger bzw. die Mikrotiterplatte mit einem primären Bindungs- partner vorbeschichtet ist und der Bindungspartner fixiert als Lyophilisat vorliegt .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.
Immuntests , Enzym gekoppelte Immuntests , Fluoreszenz Label gekoppelte Immuntests , Lumineszenz Label gekoppelte Immuntests oder mit einem anderen Label markierte Immuntests werden in der Routinediagnostik und Forschungsdiagnos tik zur Detektion und Quantifizierung von Proteinen, zum Beispiel Antigenen, Liganden, Rezeptoren, Antikörpern sowie von chemischen Verbindungen eingesetzt . Stand der Technik ist es , in solchen Test-Kits einen der Bindungspartner an einen festen Träger, zum Beispiel eine Polystyrolplatte, zu binden und die weiteren für die Versuchsdurchführung benötigten Reagentien als separate . Komponenten des Testkits hinzuzufügen . Diese Reagentien sind üblicherweise das nachzuweisende Protein in quantifizierter Form als Konzentrat oder Lyophilisat (Referenzstandard) , ein Standard- und Probenverdünnungsmedium, der zweite spezifische Bindungspartner in gekoppelter Form (Konjugat) als Konzentrat oder Arbeitslösung, falls benötigt ein sekundäres Detektionssystem sowie im Falle eines Enzymlabels das zum Nachweis benötigte Substrat-Reagenz . Zur Terminierung der enzymati sehen Reaktion wird eine Stopplösung beigefügt , zum Herstellen der Arbeitslösungen für den zweiten spezifischen Bindungspartner sowie für das Sekundärreagenz steht ein Assaypuffer zur Verfügung, welcher meist als Konzentrat vorliegt . Da zwischen den Arbeitsschritten Waschschritte erforderlich sind, enthält ein herkömmlicher Immun- Test-Kit einen Waschpuf fer , üblicherweise in konzentrierter Form.
Kommerzi elle ELISA Kits bieten dem Anwender validierte Testsysteme . Die Produkte enthalten alle für die Testdurchführung erforderlichen Reagentien in optimierten Konzentrationen und Mengen sowie ein detailliertes Protokoll zur Durchführung •des - -Tes- s -.- -Die -e-i-n-z e-l-nen- Rea-kt-i ons s c-h-r-i-t fee-we- den— üb-l-i cher-wei s e- konsekutive durchgeführt . Die gemeinsame Inkubation von Analyten ( Standard, Probe) und sekundärem Antikörper mit der Festphase (Cocktail) ist für eine Vielzahl von Testsystemen möglich.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. These 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.
Ein typisches Beispiel für einen Immuntest ist der Sandwich ELISA (Enzyme linked immunosorbent assay) .A typical example of an immunoassay is the sandwich ELISA (enzyme linked immunosorbent assay).
Typischer Weise enthal ten solche ELISA Ki ts folgende Komponente :Such ELISA kits typically contain the following components:
• Mikrotiterplatte mit 96 Vertiefungen, vorbeschichtet mit dem primären Antikörper, geblockt, fixiert .• 96-well microtiter plate, pre-coated with the primary antibody, blocked, fixed.
• Referenzmaterial : Analyt (Standard) in definierter Konzentration zur Erstellung einer Standardreihe• Reference material: analyte (standard) in a defined concentration to create a standard series
• Enzym- (Horse Radish Peroxidase) oder Biotin-gekoppelter S ekundäran ikörper• Enzyme (Horse Radish Peroxidase) or Biotin-coupled secondary body
• Falls erforderlich Streptavidin- Enzym (Horse Radish Peroxidase)• If necessary streptavidin enzyme (Horse Radish Peroxidase)
• Substratlösung (Tetramethylen-Benzidin)• substrate solution (tetramethylene benzidine)
« Waschpuffer : Salzlösung zum Waschen der Platten vor der Probeninkübation und zwischen den Reaktionsschritten«Wash buffer: saline solution for washing the plates before sample incubation and between the reaction steps
• Assay-Puf fer : Salzlösung zur Verdünnung des Sekundärantikörpers und Streptavidin-Enzym Konzentrates (Herstellen der Arbeitslösungen)• Assay buffer: saline solution for diluting the secondary antibody and streptavidin enzyme concentrate (preparation of working solutions)
• Probenverdünnungsmedium: Spezifisches flüssiges Medium zur Verdünnung des Referenzmaterials sowie der unbekannten Proben• Sample dilution medium: Specific liquid medium for dilution of the reference material and the unknown samples
• Stopp-Lösung zur Beendigung der enzymatischen Reaktion• Stop solution to stop the enzymatic reaction
Zur Testdurchführung sind durch den Anwender folgende Arbeits- schritte durchzuführen:
Herstellung der ArbeitslösungenThe user must carry out the following work steps to carry out the test: Production of working solutions
Erstellung der Standardreihe durch Verdünnung des ReferenzmaterialsCreation of the standard series by diluting the reference material
Waschen der Mikrotiterplatte -Vo-r-legen— es— P-robenver-dünnungsmedi-ums-Washing the microtiter plate -Pre-r-put— es— P-robenver-Dünnungsmedi-ums-
Auf tragen der einzelnen Referenzverdünnungen ( Standardreihe)Applying the individual reference dilutions (standard series)
Auftragen der unbekannten ProbenApplication of the unknown samples
Zugabe des Enzym- oder Biotin-gekoppelten SekundärantikörpersAddition of the enzyme or biotin-coupled secondary antibody
Waschen der Mikrotiterplatte Optional Zugabe von Streptavidin-Enzym Waschen der Mikrotiterplatte Zugabe des Substrates Zugabe der Stopplösung Messung der optischen DichteWashing the microtiter plate. Optional addition of streptavidin enzyme. Washing the microtiter plate. Adding the substrate. Adding the stop solution. Measuring the optical density
Die Erfindung zielt nun darauf ab, die Handhabung derartiger Test-Kits zu vereinfachen und neben einer qualitativen Aussage auch quantitative Aussagen zu ermöglichen . Des weiteren zielt die Erfindung neben der Reduktion des Arbeitsaufwandes und der zur Tes tdurchfühung benötigten Zeit darauf ab , die Fehlerwahrscheinlichkeit für den Anwender durch Minimierung der durchzuführenden Arbeitsschritte zu reduzieren . Schließlich ist es Ziel der vorliegenden _ Erfindung, die Verpackungsvolumina sowie Verpackungskosten ebenso wie die Versandkosten durch deutliche Gewichtsverringerung und Volums Verringerung der Kits herabzusetzen und eine logistische Vereinfachung bereitzustellen, bei welcher standardisierte Basis-Kits mit verbesserter Lagerfähigkeit des Produktes zur Verfügung gestellt werden .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. In addition to reducing the amount of work and the time required to carry out the test, the invention also aims to reduce the likelihood of errors for the user by minimizing the work steps to be carried out. Finally, 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.
Zur Lösung dieser Aufgabe besteht der erfindungsgemäße Immun- Test-Kit im wesentlichen darin, daß in einem Teil der Vertiefungen der Mikrotiterplatte zusätzlich eine Referenzstandardreihe der zu bestimmenden Probe mit inkrementeller Verdünnung in lyophilisierter Form vorliegt . Dadurch, daß der Träger bzw. die Mikrotiterplatte zusätzlich bereits eine Refe-
renzstandardreihe der zu bestimmenden Probe enthält , wird die Möglichkeit geschaffen, neben einer rein qualitativen Analyse auch eine quantitative Abschätzung und eine quantitative Bestimmung durch Interpolation zwischen definierten Testergebnissen im -Bereich -zwi-schen- -i-nk-rement-ell -benachbar-t-en— Standards- -~vor-zu-=-.. nehmen , wobei der aufwendige Schritt der Herstellung der Arbeitslösung des Ref erenzmateriales und der Herstellung der entsprechenden Verdünnungsreihe bei der Laborbestimmung entfällt . Mit einer derartigen Ausbildung des Immun-Test-Kits , bei welcher das Herstellen der Arbeitslösung des Referenzmaterials und das Auftragen des Standardverdünnungsmediums in die für die Standardreihe vorgesehenen Vertiefungen der Mikrotiterplatte entfällt , läßt sich somit bereits eine wesentliche Rationalisierung und Vereinfachung des Verfahrens und insbesondere eine exakt reproduzierbare Standardreihe vorgeben, wodurch die Fehlerwahrscheinlichkeit bereits wesentlich verringert wird.To achieve this object, 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. The fact that the carrier or the microtiter plate already has a reference contains the standard series of samples to be determined, the possibility is created, in addition to a purely qualitative analysis, also a quantitative estimate and a quantitative determination by interpolation between defined test results in the range -between- -i-nk-rement-ell -benachbar-t -en— Standards- - ~ vor-zu - = - .., the elaborate step of preparing the working solution of the reference material and the preparation of the corresponding dilution series in the laboratory determination being omitted. With such a design of the immunoassay kit, in which the preparation of the working solution of the reference material and the application of the standard dilution medium in the recesses of the microtiter plate provided for the standard series are dispensed with, a substantial rationalization and simplification of the method and in particular an exact one can already be achieved Specify reproducible standard series, which already significantly reduces the probability of errors.
In besonders vorteilhafter Weise kann aber die Vorfertigung des Immun-Test-Kits noch wesentlich weiter vorangetrieben werden und die notwendigen Bestimmungsschritte für die Probe weiter reduziert werden. Zu diesem Zweck ist mit Vorteil die Ausbildung so getroffen, daß die Vertiefungen der Mikrotiterplatte zusätzlich ein Enzym- oder Biotin-gekoppeltes Konjugat eines Sekun- därbindungspartners , insbesondere -antikörpers , und im Fall des Biotin-gekoppelten Konjugates ein Enzym in lyophilisierter Form enthalten . Auf diese Weise wird neben der Standardreihe zur Quantifizierung des nachzuweisenden Proteins in definierten Proteinkonzentrationen auch der zweite, spezifische mit einem der oben genannten Labels gekoppelte Bindungspartner (Konjugat in titrierter Konzentration sowie gegebenfalls das sekundäre Reagenz ) zum Nachweis gemeinsam mit dem fest an Phasen gebundenen primären Bindungspartner bereits in lyophilisierter Form in der Festphase eingebracht, wobei die Bereitstellung dieser Komponenten in lyophilisierter Form, d . h . somit in bei tiefen Temperaturen von etwa -30 °C dehydrierter Form, die Reaktionskinetik soweit einfriert, daß keine Vorabreaktion des Referenzmaterials zu befürchten ist . Da hochverdünnte Arbeitslösungen
ebenso wie begrenzt stabile Lösungen nicht vorrätig gehalten werden, sondern vielmehr erst unmittelbar zu Testbeginn durch Zugabe von Lösungsmittel bzw. Rehydrierung auf dem Träger erzeugt werden, werden weitere mögliche Fehlerquellen ausge- -schlossen.-In a particularly advantageous manner, however, the prefabrication of the immunoassay kit can be pushed much further and the necessary determination steps for the sample can be further reduced. For this purpose, 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. In this way, in addition to the standard series for the quantification of the protein to be detected in defined protein concentrations, 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.
In weiterer Verbesserung und zur weiteren Verringerung der erforderlichen Arbeitsschritte bei der Bestimmung ist die Ausbildung so getroffen, daß die Vertiefungen der Mikrotiterplatte ein Probenverdünnungsmedium in lyophilisierter Form enthalten. Das spezifische Probenverdünnungsmedium kann hiebei bei einer Temperatur von beispielsweise 2°C in der benötigten Menge in die entsprechenden Vertiefungen der Mikrotiterplatte eingebracht werden, wobei das Sekundärantikörper-Enzymkonjugat bzw. das Gemisch aus Sekundärantikörper-Biotinkonjugat bei gleicher Temperatur in alle Vertiefungen der Mikrotiterplatte eingebracht werden kann, worauf die so beschickte Mikrotiterplatte einem Gefriertrocknungsprozeß in einer beispielsweise auf -30°C vorgekühlten Gefriertrocknungsanlage der Lyophilisierung unterzogen wird.In a further improvement and to further reduce the necessary work steps in the determination, 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.
Ein derartiger vorkonfektionierter Träger bzw. eine derartige vorkonfektionierte Mikrotiterplatte erfordert zur Vervollständigung des Test-Kits nur mehr eine geringe Anzahl zusätzlicher Komponenten, wobei mit Vorteil der Test-Kit zusätzlich zur vorbeschichteten Mikrotiterplatte lediglich Waschpuffer, Substratlösung und Stopplösung enthält. Für die Bestimmung der Probe ist es somit lediglich erforderlich, die Mikrotiterplatte durch Zugabe definierter Volumina an destilliertem Wasser zu den Vertiefungen der Standardreihe, gegebenenfalls den Blanks und den Probenvertiefungen, zu rehy- drieren, worauf die unbekannte Probe aufgegeben und inkubiert wird. Nach einem Waschen der Mikrotiterplatte und dem Auftragen des Substrates in alle für den Test verwendeten Vertiefungen der Mikrotiterplatte wird nach vorbestimmter Zeit die Stopplösung zugegeben und es kann unmittelbar die Testauswertung vorgenommen werden.
Mit Vorteil ist die Ausbildung hiebei so getroffen, daß im Falle von Biotin-gekoppeltem Sekundärantikörper-Konjugat als Sekundärreagenz Streptavidin - Horse Radish Peroxidase (HRP) eingesetzt ist-.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. 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. After washing the microtiter plate and applying the substrate in all wells of the microtiter plate used for the test, 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.
Insgesamt wird durch die erfindungsgemäße Ausbildung die Testdurchführung für den Anwender auf die Zugabe der unbekannten Probe sowie die enzymatische Reaktion reduziert, wobei alle weiteren Schritte zuvor vom Hersteller des ELISA Kits durchgeführt werden. Die Mikrotiterplatte wird somit beim Hersteller mit dem spezifischen Primärantikörper beschichtet, geblockt und fixiert, worauf die Platte beispielsweise auf -20°C gekühlt wird und die oben beschriebenen zusätzlichen Schritte zum Einbringen der Standardreihe des Probenverdünnungsmittels sowie des Sekun- därantikörper-Enzymkonjugates bzw. des Gemisches aus Sekundär- antikörper-Biotinkonjugates und Streptavidin-Enzymes in definierter Weise beim Hersteller vorgenommen werden. Die Testdurchführung reduziert sich somit auf die Zugabe der zu analysierenden Proben sowie die Testauswertung aufgrund der Substratfärbung.Overall, the 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.
In besonders vorteilhafter Weise ist der Immun-Test-Kit nach der vorliegenden Erfindung so ausgestaltet, dass der Test-Kit Stabilisatoren für Peroxidase beziehungsweise generelle Lyoprotektanten wie Proteine (z.B.Albumin (z.B. BSA) Protein Hydrolysate (Peptone, Gelatine...) Casein) , Polymeren (z.B. Dextran, PVA, PVP) , Zucker (z.B. Saccharose, Trehalose, Lactose, Xylit, Sorbit, Mannit, Maltose, Glucose, Inosit), Bakteriostatische Mittel (z.B. Thimerosal, Proclin) , phenolische Substanzen und Aniline auch mit Substituenten (kleine Alkyl- Reste oder Cl, Br...) (z. B. o-Methoxyphenol, o-Methylphenol, p- Methylphenol, o-Aminophenol, o-Hydroxybenzoesaäure, (o, m oder p) -Hydroxybenzylalkohol, Anilin, p-Aminobenzoesäure, p-Methoxy- anilin, Benzylalkohol, Benzoesäure, p-Nitrophenol, Benzylamin, 1-Phenyl-l, 2-ethandiol, trans-1, 2-Cyclohexandiol, cis-1, 2-Cyclo- hexandicarbonsäure, Cyclohexylamin) ; Hydrophobe Verbindungen und
Verbindungen und Lösungsmitteln (z.B. DMF, Ethylenglykol, DMSO) ; Detergenzien (z.B. Tween-20); Aryl-Borsäureverbindungen (z.B. Phenylborsäure, 4-Brom-Phenylborsäure, 3-In a particularly advantageous manner, 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. thimerosal, proclin), 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, 4-bromo-phenyl boric acid, 3-
Acetamidophenyl—borsäure, 1-Naphtyl—borsäure) ; Substratanalogen (z.B. TMB, Luminol) ; Poly-Hydroxy-Verbindungen (z.B. Polyole, Polyethylenglykol, Glyzerin); Ectoine (z.B. (S)-2-methyl- 1,4,5, 6-tetrahydropyrimidine-4-carbo—ylicacid [ HP(B) ] , (S,S ) - ß-2-methyl-5-hydroxy-l,2,4,5,6- tetra—hydropyrimidine-4- carboxylic-acid, [THP(A)]); Ionen bzw. polyvalente Ionen (z. B. Metalionen (AI, Zn, Mg, Fe, Cu,...)); Komplexbildnern (z.B. EDTA) ; Aminosäuren (z.B. Glyzin, Prplin, 4-Hydroxyprolin, Serin, Glutamat, Alanin, Lysin, Sarcosin, y-Aminobutyruc acid, Phenylalani ) und/oder Substanzen wie TRIS, Salze, Aine, Na- Cholate, Saccharose monolaurat, 2-O-ß-Mannosylglyzerat enthält.Acetamidophenyl boric acid, 1-naphthyl boric acid); Substrate analogs (e.g. 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 (B)], (S, S) - β-2-methyl-5-hydroxy-l , 2,4,5,6-tetra-hydropyrimidine-4-carboxylic acid, [THP (A)]); Ions or polyvalent ions (e.g. 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) and / or substances such as TRIS, salts, aines, Na cholates, sucrose monolaurate, 2-O- contains β-mannosylglycerate.
Mit Rücksicht auf die besonders einfache Verfahrensweise eignet sich der vorliegende Immuntest-Kit für eine Vielzahl von möglichen Anwendungen. In besonders vorteilhafter Weise erfolgt die Verwendung eines derartigen Immuntest-Kits für den Einsatz in der Forschungsdiagnostik und in-vitro Diagnostik, für den Einsatz in der ErregerSerologie, wie zum Beispiel zum Nachweis von Infektionen durch HIV, HepV , EBV, CMV usw. , für den Einsatz in der Tumordiagnostik, wie zum Beispiel zum Nachweis von Tumormarkern oder Tumor assoziierten Proteinen, Vaskula—risierungsmar ern, Metastasierungsmarkern usw. , für den Einsatz in der Allergologie wie zum Beispiel zum Nachweis von Tierallergenen, Pflanzenpollen, Nahrungsbestandteilen usw., für den Einsatz in der Autoi mun-Diagnostik wie zum Beispiel zum Nachweis der Auto-Antigene im Bereich ANA/ENA, Diabetes mellitus/ IDDM, Schilddrüsen Antigene, Autoimmun Hepatitis, APS, usw. , für den Nachweis von immunologischen Störungen durch entzündliche Prozesse oder Infektionen wie zum Beispiel zum Nachweis von Zytokinen, Adhäsionsmolekülen, Chemokinen, usw. , für den Nachweis von Änderungen des Hormonhaushaltes wie zum Beispiel Ovulationstests, Schwangerschaftstests, usw., für den Einsatz zum Therapie-Monitoring wie zum Beispiel zum Nachweis von therapeutischen Antikörpern und Antigenen, organischen und
anorganischen Verbindungen, für den Nachweis von Zelltod durch Apoptose oder Nekrose und/oder für den Nachweis von genetischen Veränderungen, genetischen Erkrankungen.In view of the particularly simple procedure, 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. for use in autoimmune diagnostics such as for the detection of autoantigens in the area of ANA / ENA, diabetes mellitus / IDDM, thyroid antigens, autoimmune hepatitis, APS, etc., for the detection of 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., for use in 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.
-Die -Erfindung -wi-rd— achfolgend -anhand -einer -Gegenüber s-tel-1-ung- zum Stand der Technik näher erläutert , wobei der Stand der Technik als Standard-Kit bezeichnet wird.The invention -wi-rd - subsequently - with reference to a - compared to s-tel-1-ung- explained in more detail with respect to the prior art, the prior art being referred to as the standard kit.
1. Komponenten der ELISA Kits :1. Components of the ELISA kits:
a) direkt Enzym gekoppeltes Konjugata) Direct enzyme-coupled conjugate
- -7- -7
b) zweiter Antikörper an Biotin gekoppelt , Sekundär reagenz Streptavidin-Horse Radish Peroxidase (HRP) o . ä .b) second antibody coupled to biotin, secondary reagent streptavidin-Horse Radish Peroxidase (HRP) o. Ä.
2. Arbeitsschritte zur Testdurchführung i2. Steps for performing the test i
a) direkt Enzym gekoppeltes Konjugata) Direct enzyme-coupled conjugate
b) zweiter Antikörper an Biotin gekoppelt, Sekundärreagenz Streptavidin-Horse Radish Peroxidase (HRP) o.ä.b) second antibody coupled to biotin, secondary reagent streptavidin-Horse Radish Peroxidase (HRP) or similar
Die Erfindung kann hiebei ihre Anwendung zur Detektion von Antigenen mit Hilfe eines Paares von Antikörper (Sandwich ELISA) finden. Ebenso ist der Nachweis von Antikörpern mit dem entsprechenden Antigen möglich. Die Detektion von Rezeptoren bzw. Liganden durch den j eweiligen Bindungspartner ist eine weitere Möglichkeit der Anwendung der vorliegenden Erfindung, sodaß eine große Anzahl von Testformaten zur Verfügung' steht . Insbesondere kann der Test zum Nachweis von Proteinen, Steroiden, chemischen Verbindungen, Medikamenten, Nukleinsäuren, und ähnlichen Substanzen eingesetzt werden.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. In particular, the test can be used to detect proteins, steroids, chemical compounds, drugs, nucleic acids, and similar substances.
Die Detektion des Reaktions komplexes kann mit Hilfe eines direkt gekoppelten Enzymes erfolgen ( zum Beispiel Horse Radish Peroxidase, Alkalische Phosphatase u. a. ) , über eine enzymatische Reaktion, welche durch einen Sekundärschritt erfolgt (Biotin- Streptavidin-HRP, enzym-gekoppelter Antikörper gegen den Detek- tionsantikörper u . a . ) . Jedes weitere Label , welches für die Durchführung eines Immuntestes geeignet ist , wie Fluorochrome, Chemiluminenszenz Labels , radioaktive Labels , u. a . , kann ebenso benutzt werden.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). 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.
Der Träger zur Immobilisierung des primären Bindungspartners ist vorzugsweise eine Polystyrol 96 Well Mikrotiterplatte, wobei j edoch j eder andere Träger, der für die Durchführung eines Immuntestes geeignet ist, ebenso für die vorliegende Erfindung eingesetzt werden kann.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.
Die zur Messung eingesetzten Proben können alle flüssigen Proben, die den Analyten enthalten, im speziellen Körperflüssigkeiten wie Seren, Plasmapräparationen, lokale Körper-
flüssigkeiten, Vollblut, u.a. sowie Zellkulturüberstände, gepufferte Lösungen der Analyten u.a., sein.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.
Die Erfindung wird nachfolgend anhand von spezifischen Anwen- -dungsbeispielen -näher er-läutert.The invention is explained in more detail below on the basis of specific application examples.
Anwendungsbeispiel 1Application example 1
Sandwich ELISA zum Nachweis von humanem ICAM-1Sandwich ELISA for the detection of human ICAM-1
A) Herstellung der Platten in Verbindung mit Proben verdünnungsmedium, Standardreihe, HRP-KonjugatA) Preparation of the plates in connection with sample dilution medium, standard series, HRP conjugate
Schritt 1 : Beschichtung:Step 1: coating:
Die 96 well Mikrotiterplatten werden nach dem Stand der Technik mit 2,5μg/ml anti-ICAM-1 in PBS Puffer beschichtet (lOOμl pro well, über Nacht Inkubation bei 4°C) . Schritt 2: Blockierung:The 96-well microtiter plates are coated according to the prior art with 2.5 μg / ml anti-ICAM-1 in PBS buffer (100 μl per well, incubation overnight at 4 ° C.). Step 2: blocking:
Die Beschichtungslösung wird abgesaugt, die Platte wird einmal mit Waschpuffer gewaschen (PBS/Tween) . Zur Absättigung der Polystyroloberfläche (Verhinderung unspezifischer Bindungen) wird die Platte mit 300μl pro well PBS/2% BSA blockiert (2h, RT) . Schritt 3 : Fixierung:The coating solution is suctioned off, the plate is washed once with washing buffer (PBS / Tween). To saturate the polystyrene surface (to prevent unspecific binding), the plate is blocked with 300μl per well PBS / 2% BSA (2h, RT). Step 3: fixation:
Die Blockierungslösung wird abgesaugt, die Platte zweimal gewaschen, pro Well werden 150μl PBS/15% Sucrose hinzugefügt. Nach einer Stunde Inkubation bei RT wird die Fixierungslösung dekantiert. Die Platte wird ca. 20 Stunden bei 28°C im U luft- trockenschrank getrocknet. Die Platte wird auf -20°C eingefroren. Schritt 4 :The blocking solution is suctioned off, the plate is washed twice, 150 μl PBS / 15% sucrose are added per well. After an hour of incubation at RT, the fixation solution is decanted. The plate is dried for approx. 20 hours at 28 ° C in an air drying cabinet. The plate is frozen to -20 ° C. Step 4:
Einbringung des Probenverdünnungsmediums :Introduction of the sample dilution medium:
Die beschichtete Platte wird im gefrorenen Zustand eingesetzt. Das Probenverdünnungsmedium wird auf 2°C gekühlt. Zur Erstellung der Standardreihe wird in den ersten beiden Reihen der Mikrotiterplatte in jede Vertiefung lOOμl Probenverdünnungsmedium
vorgelegt . Für die Bestimmung des Blanks wird in die entsprechenden Vertiefungen lOOμl Probenverdünnungsmedium vorgelegt . Zur Bestimmung der unbekannten Proben wird in j ede entsprechende Vertiefung 90μl Probenverdünnungsmedium vorgelegt . -Schri-t —5 :The coated plate is used in the frozen state. The sample dilution medium is cooled to 2 ° C. To create the standard series, 100 μl sample dilution medium is placed in each well in the first two rows of the microtiter plate submitted . To determine the blank, 100 μl sample dilution medium is placed in the corresponding wells. To determine the unknown samples, 90 μl sample dilution medium is placed in each corresponding well. - Step -5:
Erstellung der Standardreihe :Creation of the standard series:
Die Arbeitlösung (20ng/ml) des Referenzmaterials (rekombinantes humanes ICAM-1 ) ( 2 °C) wird in Doppelbestimmung in die beiden obersten Vertiefungen auf der linken Seite der Mikrotiterplatte eingebracht (lOOμl/well ) . Durch serielle 1 : 2 Verdünnung in der Platte wird eine Standardreihe hergestellt ( lOng/ml ; 5ng/ml ; 2 , 5ng/ml ; l, 25ng/ml ; 0 , 63ng/ml) . Schritt 6:The working solution (20ng / ml) of the reference material (recombinant human ICAM-1) (2 ° C) is introduced in duplicate into the two uppermost wells on the left side of the microtiter plate (100 μl / 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). Step 6:
Einbringung des HRP-Kon jugates :Introduction of the HRP conjugate:
Die Arbeitslösung des HRP-Kon jugates (anti-ICAM-1-HRP) wird auf 2°C gekühlt und rasch in alle Vertiefungen der Mikrotiterplatte eingebracht (50μl/well) . Schritt 7: Lyophilisierung:The working solution of the HRP conjugate (anti-ICAM-1-HRP) is cooled to 2 ° C and quickly introduced into all wells of the microtiter plate (50μl / well). Step 7: lyophilization:
Die Mikrotiterplatte wird unmittelbar nach Beschickung mit allen Komponenten mit einer Folie abgedeckt und in die auf -30 °C vorgekühlte Gefriertrocknungsanlage eingebracht . Die Lyophilisierung erfolgt ca. 20 Stunden bei -30°C . Die getrocknete Platte wird unmittelbar nach der Entnahme aus der Gefriertrocknungsanlage zusammen mit Trocknungsmittel in eine Aluminiumtasche eingeschweißt .Immediately after loading, 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.
B ) Tes t dur chf ührung :B) Test execution:
Die Platte wird aus der Aluminiumtasche genommen. Die Standardreihe sowie die Blanks werden durch Zugabe von 150μl destilliertem Wasser pro Vertiefung rehydriert , die Probenvertiefungen durch Zugabe von 140μl H20. In die Probenvertiefungen werden j e lOμl der unbekannten Probe aufgetragen. Die Platte wird mit einer Folie abgedeckt und 1 Stunde bei RT inkubiert . Die Platte wird 3mal gewaschen, in jede Vertiefung der Platte wird lOOμl Substratlösung zugegeben. Die Enzymreaktion wird nach
15 Minuten durch die Zugabe der Stopplösung ( lOOμl ) abgebrochen und die Farbintensität in den einzelnen Vertiefungen photometrisch ausgewertet .The plate is taken out of the aluminum bag. The standard series and the blanks are rehydrated by adding 150μl distilled water per well, the sample wells by adding 140μl H20. 10 μl 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 μl 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 μl) and the color intensity in the individual wells was evaluated photometrically.
Anwendungsbeispiel 2Example of use 2
Sandwich ELISA zum Nachweis von humanem nterleu in 10Sandwich ELISA for the detection of human nterleu in 10
( IL-10 )(IL-10)
A) Herstellung der Platten in Verbindung mit Probenverdünnungsmedium, Standardreihe, Biotin-Konjugat , Streptavidin-HRPA) Preparation of the plates in connection with sample dilution medium, standard series, biotin conjugate, streptavidin-HRP
Schritt 1 : Bes chi chtung :Step 1: review:
Die 96 well Mikro titerplatten werden nach dem Stand der Technik mit 5μg/ml anti-IL-10 in PBS Puffer beschichtet (lOOμl pro well , über Nacht Inkubation bei 4°C) . Schritt 2: Blockierung:The 96-well micro titer plates are coated according to the prior art with 5μg / ml anti-IL-10 in PBS buffer (100μl per well, incubation overnight at 4 ° C.). Step 2: blocking:
Die Beschichtungslösung wird abgesaugt, die Platte wird einmal mit Waschpuffer gewaschen (PBS/Tween) . Zur AbSättigung der Polystyroloberfläche (Verhinderung unspezifischer Bindungen) wird die Platte mit 300μl pro well PBS/2% BSA blockiert (2h, RT) . Schri tt J .Fixierung:The coating solution is suctioned off, the plate is washed once with washing buffer (PBS / Tween). To saturate the polystyrene surface (to prevent unspecific binding), the plate is blocked with 300μl per well of PBS / 2% BSA (2h, RT). Step J. Fixation:
Die Blockierungslösung wird abgesaugt , die Platte zweimal gewaschen, pro Well werden 150μl PBS/15% Sucrose hinzugefügt . Nach einer Stunde Inkubation bei RT wird die Fixierungslösung dekantiert . Die Platte wird ca . 20 Stunden bei 28 °C im Umluft- trockenschrank getrocknet . Die Platte wird auf -20 °C eingefroren . Schritt 4 :The blocking solution is suctioned off, the plate is washed twice, 150 μl PBS / 15% sucrose are added per well. After an hour of incubation at RT, the fixation solution is decanted. The plate is approx. Dried for 20 hours at 28 ° C in a convection oven. The plate is frozen to -20 ° C. Step 4:
Einbringung des Probenverdünnungsmediums :Introduction of the sample dilution medium:
Die beschichtete Platte wird im gefrorenen Zustand eingesetzt . Das Probenverdünnungsmedium wird auf 2°C gekühlt . Zur Erstellung der Standardreihe wird in den ersten beiden Reihen der Mikrotiterplatte in j ede Vertiefung lOOμl Pr obenver dünnungs -
medium vorgelegt. Für die Bestimmung des Blanks wird in die entsprechenden Vertiefungen lOOμl Probe Verdünnungsmedium vorgelegt. Zur Bestimmung der unbekannten Proben wird in jede Vertiefung 50μl Probenverdünnungsmedium vorgelegt. -Sσhri tt- S :■The coated plate is used in the frozen state. The sample dilution medium is cooled to 2 ° C. To create the standard series, the first two rows of the microtiter plate are diluted with 100 μl Pr in each well - medium presented. For the determination of the blank, 100 μl sample of dilution medium is placed in the corresponding wells. To determine the unknown samples, 50μl sample dilution medium is placed in each well. -Sσhri tt- S: ■
Erstellung der Standardreihe:Creation of the standard series:
Die Arbeitlösung (400pg/ml) des Referenzmaterials (rekombinantes humanes IL-10) (2°C) wird in Doppelbestimmung in die beiden obersten Vertiefungen auf der linken Seite der Mikrotiterplatte eingebracht (lOOμl/well) . Durch serielle 1:2 Verdünnung in der Platte wird eine Standardreihe hergestellt (200 - 3.1 pg/ml) . Schri tt 6: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 (100μl / well). A standard series (200-3.1 pg / ml) is prepared by serial 1: 2 dilution in the plate. Step 6:
Einbringung des Biotin-Ko jugates und Streptavidin-HRP: Die Arbeitslösung des Gemisches aus Biotin-Konjugat (anti-IL-10- BT) und Streptavidin-HRP wird auf 2°C gekühlt und rasch in alle Vertiefungen der Mikrotiterplatte eingebracht (50μl/well) . Schritt 7: Lyophilisierung:Introduction of the biotin conjugate and streptavidin-HRP: The working solution of the mixture of biotin conjugate (anti-IL-10-BT) and streptavidin-HRP is cooled to 2 ° C and quickly introduced into all wells of the microtiter plate (50μl / well ). Step 7: lyophilization:
Die Mikrotiterplatte wird unmittelbar nach Beschickung mit allen Komponenten mit einer Folie abgedeckt und in die auf -30°C vorgekühlte Gefriertrocknungsanlage eingebracht. Die Lyophilisierung erfolgt ca. 20 Stunden bei -30°C. Die getrocknete Platte wird unmittelbar nach der Entnahme aus der Gefriertrocknungs- anlage zusammen mit Trocknungsmittel in eine Aluminiumtasche eingeschweißt.Immediately after loading, 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.
B) Testdurchführung:B) Test execution:
Die Platte wird aus der Aluminiumtasche genommen. Die Standardreihe sowie die Blanks werden durch Zugabe von 150μl destilliertem Wasser pro Vertiefung rehydriert, die Probenvertiefungen durch Zugabe von lOOμl H20. In die Probenvertiefungen werden je 50μl der unbekannten Probe aufgetragen. Die Platte wird mit einer Folie abgedeckt und 3 Stunden bei RT inkubiert. Die Platte wird 3mal gewaschen, in jede Vertiefung der Platte wird lOOμl Substratlösung zugegeben. Die Enzymreaktion wird nach 15 Minuten durch die Zugabe der Stopplösung
( l O Oμl ) abgebrochen und die Farbintensität in den einzelnen Vertiefungen photometrisch ausgewertet .The plate is taken out of the aluminum bag. The standard series and the blanks are rehydrated by adding 150μl distilled water per well, the sample wells by adding lOOμl H20. 50μl 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 μl 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 Oμl) canceled and the color intensity in the individual wells was evaluated photometrically.
Anwendungsbeispiel 3Example of use 3
-Inver-ser-- Sandwic — ELISA zum - -Nachweis — on — umanem- Antikörpern gegen Interferon alpha (IFNOt)-Inver-ser-- Sandwic - ELISA for - detection - on - umanem- antibodies against interferon alpha (IFNOt)
A) Herstellung der Platten in Verbindung mit Probenverdünnungsmedium, Standardreihe, HRP-KonjugatA) Preparation of the plates in connection with sample dilution medium, standard series, HRP conjugate
Schritt 1 :Step 1 :
Beschichtung: Die 96 well Mikrotiterplatten werden nach dem Stand der Technik mit lOμg/ml Streptavidin in PBS Puffer beschichtet (lOOμl pro well, über Nacht Inkubation bei 4°C) . Schritt 2 :Coating: The 96-well microtiter plates are coated according to the prior art with 10 μg / ml streptavidin in PBS buffer (100 μl per well, incubation overnight at 4 ° C.). Step 2 :
Spezifische Beschichtung/Blockierung: Die Streptavidin-Beschich- tungslösung wird abgesaugt, die Platte wird einmal mit Waschpuffer gewaschen (PBS/Tween) . Zur spezifischen Beschichtung der Platte sowie zur AbSättigung der Polystyroloberfläche (Verhinderung unspezifischer Bindungen) wird die Platte mit 300μl pro well IFNα-Biotin Konjugat, lμg/ l in PBS/2% BSA beschichtet beziehungsweise blockiert (2h, 37°C) .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 300μl per well IFNα-biotin conjugate, lμg / 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).
Schritt 3 :Step 3 :
Fixierung: Die Beschichtungs/Blockierungslösung wird abgesaugt, die Platte zweimal gewaschen, pro Well werden 150μl PBS/15%Fixation: The coating / blocking solution is suctioned off, the plate is washed twice, 150μl PBS / 15% are used per well
Sucrose hinzugefügt. Nach einer Stunde Inkubation bei RT wird die Fixierungslösung dekantiert. Die Platte wird ca. 20 Stunden bei 28°C im Umlufttrockenschrank getrocknet. Die Platte wird aufSucrose added. After an hour of incubation at RT, the fixation solution is decanted. The plate is dried for approx. 20 hours at 28 ° C in a forced air drying cabinet. The plate is on
-20°C eingefroren.Frozen at -20 ° C.
Schritt 4 :Step 4:
Einbringung des Probenverdünnungsmediums :Introduction of the sample dilution medium:
Die beschichtete Platte wird im gefrorenen Zustand eingesetzt.The coated plate is used in the frozen state.
Das Probenverdünnungsmedium wird auf 2°C gekühlt. Zur Erstellung der Standardreihe wird in den ersten beiden Reihen derThe sample dilution medium is cooled to 2 ° C. To create the standard series, the
Mikrotiterplatte in jede Vertiefung lOOμl Probenverdünnungs- edium vorgelegt. Für die Bestimmung des Blanks wird in die entsprechenden Vertiefungen lOOμl Probenverdünnungsmedium vor-
gelegt. Zur Bestimmung der unbekannten Proben wird in jede Vertiefung 75μl Probenverdünnungsmedium vorgelegt. Schri tt S.Erstellung der Standardreihe:Place microtiter plate in each well of 100 μl sample dilution medium. For the determination of the blank, 100 μl sample dilution medium is prepared in the corresponding wells. placed. To determine the unknown samples, 75μl sample dilution medium is placed in each well. Step S. Creation of the standard series:
-Die Arbeitlösung- -(-2-0Ong/-ml) des -Ref.erenzmater als -(-anti—human- IFNα Antikörper) (2°C) wird in Doppelbestimmung in die beiden obersten Vertiefungen auf der linken Seite der Mikrotiterplatte eingebracht (lOOμl/well) . Durch serielle 1:,2 Verdünnung in der-The working solution- - (- 2-0Ong / -ml) of the -Ref.erenzmater as - (- anti-human- IFNα antibody) (2 ° C) is introduced in duplicate in the two top wells on the left side of the microtiter plate (100μl / well). By serial 1:, 2 dilution in the
Platte wird eine Standardreihe hergestellt (100 - l,6ng/ml).Plate is made a standard series (100 - 1, 6ng / ml).
Schritt 6:Step 6:
Einbringung des HRP-Konjugates :Introduction of the HRP conjugate:
Die Arbeitslösung des HRP-gekoppelten IFNα Proteins wird auf 2°C gekühlt und rasch in alle Vertiefungen der Mikrotiterplatte eingebracht (50μl/well) . Schritt 7: Lyophi1isierung:The working solution of the HRP-coupled IFNα protein is cooled to 2 ° C and quickly introduced into all wells of the microtiter plate (50μl / well). Step 7: lyophilization:
Die Mikrotiterplatte wird unmittelbar nach Beschickung mit allen Komponenten mit einer Folie abgedeckt und in die auf -30°C vorgekühlte Gefriertrocknungsanlage eingebracht. Die Lyophili- sierung erfolgt ca. 20 Stunden bei -30°C. Die getrocknete Platte wird unmittelbar nach der Entnahme aus der Gefriertrocknungs- anlage zusammen mit Trocknungsmittel in eine Aluminiumtasche eingeschweißt.Immediately after loading, 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.
B) Testdurchführung:B) Test execution:
Die Platte wird aus der Aluminiumtasche genommen. Die Standardreihe sowie die Blanks werden durch Zugabe von 150μl destilliertem Wasser pro Vertiefung rehydriert, die Probenvertiefungen durch Zugabe von 125μl H20.In die Probenvertiefungen werden je 25μl der unbekannten Probe aufgetragen. Die Platte wird mit einer Folie abgedeckt und 2 Stunden bei RT inkubiert.Die Platte wird 3mal gewaschen, in jede Vertiefung der Platte wird lOOμl Substratlösung zugegeben. Die Enzymreaktion wird nach 15 Minuten durch die Zugabe der Stopplösung (lOOμl) abgebrochen und die Farbintensität in den einzelnen Vertiefungen photometrisch ausgewertet.
Anwendungsbeispiel 4The plate is taken out of the aluminum bag. The standard series and the blanks are rehydrated by adding 150μl distilled water per well, the sample wells by adding 125μl H20. 25μl 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 μl 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 μl) and the color intensity in the individual wells is evaluated photometrically. Example of use 4
BioLISA zum Nachweis von humanem Tumor Necrosis Factor alpha (TNFα) (Rezeptor-Liganden Bindung)BioLISA for the detection of human tumor necrosis factor alpha (TNFα) (receptor-ligand binding)
A) -Hers-teilung -der -Platten in— Verbindung -mit -Probenverdünnungsmedium, Standardreihe, Biotin-Konjugat , Streptavidin-HRPA) -Distribution of plates in conjunction with sample dilution medium, standard series, biotin conjugate, streptavidin-HRP
Schritt 1 : Beschichtung :Step 1: coating:
Die 96 well Mikrotiterplatten werden nach dem Stand der Technik mit lμg/ml rekombinantem TNF-Rezeptor in PBS Puffer beschichtet (lOOμl pro well , über Nacht Inkubation bei 4°C) . Schritt 2: Blockierung:The 96-well microtiter plates are coated according to the prior art with 1 μg / ml recombinant TNF receptor in PBS buffer (100 μl per well, overnight incubation at 4 ° C.). Step 2: blocking:
Die Beschichtungslösung wird abgesaugt, die Platte wird einmal mit Waschpuffer gewaschen (PBS/Tween) . Zur Absättigung der Polystyroloberfläche (Verhinderung unspezifischer Bindungen) wird die Platte mit 300μl pro well PBS/2% BSA blockiert (2h, RT) . Schritt 3 : Fixierung:The coating solution is suctioned off, the plate is washed once with washing buffer (PBS / Tween). To saturate the polystyrene surface (to prevent unspecific binding), the plate is blocked with 300μl per well PBS / 2% BSA (2h, RT). Step 3: fixation:
Die Blockierungslösung wird abgesaugt, die Platte zweimal gewaschen, pro Well werden 150μl PBS/15% Sucrose hinzugefügt . Nach einer Stunde Inkubation bei RT wird die Fixierungslösung dekantiert . Die Platte wird ca . 20 Stunden bei 28 °C im Umluf t- trockenschrank getrocknet . Die Platte wird auf -20 °C eingefroren. Schritt 4 :The blocking solution is suctioned off, the plate is washed twice, 150 μl PBS / 15% sucrose are added per well. After an hour of incubation at RT, the fixation solution is decanted. The plate is approx. Dried for 20 hours at 28 ° C in a circulating air drying cabinet. The plate is frozen to -20 ° C. Step 4:
Einbringung des Probenverdünnungsmediums :Introduction of the sample dilution medium:
Die beschichtete Platte wird im gefrorenen Zustand eingesetzt . Das Probenverdünnungsmedium wird auf 2 °C gekühlt . Zur Erstellung der Standardreihe wird in den ersten beiden Reihen der Mikrotiterplatte in j ede Vertiefung lOOμl Probenverdünnungs- medium vorgelegt . Für die Bestimmung des Blanks wird in die entsprechenden Vertiefungen lO Oμl Probenverdünnungsmedium vorgelegt . Zur Bestimmung der unbekannten Proben wird in j ede Vertiefung 50μl Probenverdünnungsmedium vorgelegt .
Schritt 5:The coated plate is used in the frozen state. The sample dilution medium is cooled to 2 ° C. To create the standard series, 100 µl sample dilution medium is placed in each well in the first two rows of the microtiter plate. To determine the blank, 10 Oμl sample dilution medium is placed in the appropriate wells. To determine the unknown samples, 50μl sample dilution medium is placed in each well. Step 5:
Erstellung der Standardreihe :Creation of the standard series:
Die Arbeitlösung (2000pg/ml ) des Referenzmaterials ( rekombi- nantes humanes TNFα) (2 °C) wird in Doppelbestimmung in dieThe working solution (2000pg / ml) of the reference material (recombinant human TNFα) (2 ° C) is duplicated in the
-beiden— obersten -Vertiefungen -auf— er — linke— Seite— der- -JYEikro--^— titerplatte eingebracht (lOOμl/well ) . Durch serielle 1 : 2 Verdünnung in der Platte wird eine Standardreihe hergestellt (1000 - 16 pg/ml) Schritt 6:- Both topmost recesses - on the left side of the - JYEikro - ^ - titer plate introduced (100μl / well). A standard series is prepared by serial 1: 2 dilution in the plate (1000 - 16 pg / ml) Step 6:
Einbringung des Biotin-Kon jugates und Streptavidin-HRP : Die Arbeitslösung des Gemisches aus Biotin-Kon jugates (anti- TNFα-BT) und Streptavidin-HRP wird auf 2°C gekühlt und rasch in alle Vertiefungen der Mikrotiterplatte eingebracht (50μl/well) . Schritt 7: Lyophi 1 i s i er ung :Introduction of the biotin conjugate and streptavidin-HRP: The working solution of the mixture of biotin conjugate (anti-TNFα-BT) and streptavidin-HRP is cooled to 2 ° C and quickly introduced into all wells of the microtiter plate (50μl / well) , Step 7: Lyophi 1 i s i er ung:
Die Mikrotiterplatte wird unmittelbar nach Beschickung mit allen Komponenten mit einer Folie abgedeckt und in die auf -30 °C vorgekühlte Gefriertrocknungsanlage eingebracht . Die Lyophili- sierung erfolgt ca. 20 Stunden bei -30°C . Die getrocknete Platte wird unmittelbar nach der Entnahme aus der Gefriertrocknungsanlage zusammen mit Trocknungsmittel in Aluminiumtaschen eingeschweißt .Immediately after loading, 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.
B ) Tes t dur chf ührung :B) Test execution:
Die Platte wird aus der Aluminiumtasche genommen. Die Standardreihe sowie die Blanks werden durch Zugabe von 150μl destilliertem Wasser pro Vertiefung rehydriert, die Probenvertiefungen durch Zugabe von lOOμl H20. In die Probenvertiefungen werden j e 50μl der unbekannten Probe aufgetragen. Die Platte wird mit einer Folie abgedeckt und über Nacht bei 4°C inkubiert . Die Platte wird 3mal gewaschen, in j ede Vertiefung der Platte wird lOOμl Substratlösung zugegeben. Die Enzymreaktion wird nach 15 Minuten durch die Zugabe der Stopplösung (lOOμl) abgebrochen und die Farbintensität in den einzelnen Vertiefungen photometrisch ausgewertet .
The plate is taken out of the aluminum bag. The standard series and the blanks are rehydrated by adding 150μl distilled water per well, the sample wells by adding lOOμl H20. 50 μl 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 μl 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 μl) and the color intensity in the individual wells is evaluated photometrically.
Claims
1 . Immun-Test-Kit für die qualitative und quantitative Bestimmung einer Probe durch spezifische Bindungspartner wie -z .-B . --Antikörper , Antigene, -Rezeptoren— und -Liganden-,-— it— einer- Träger- bzw. einer Mikrotiterplatte mit einer Mehrzahl von Vertiefungen, wobei der' Träger bzw. die Mikrotiterplatte mit einem primären Bindungspartner vorbeschichtet ist und der Bindungspartner fixiert als Lyophilisat vorliegt , dadurch gekennzeichnet, daß in einem Teil der Vertiefungen der Mikrotiterplatte zusätzlich eine Referenzstandardreihe der zu bestimmenden Probe mit inkrementeller Verdünnung in lyophilisierter Form vorliegt .1 . Immune test kit for the qualitative and quantitative determination of a sample by specific binding partners such as -z.-B. - Antibodies, antigens, receptors and ligands, a carrier or a microtiter plate with a plurality of wells, the carrier or the microtiter plate being precoated with a primary binding partner and the binding partner being fixed is present as a lyophilisate, characterized 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.
2. Immun-Test-Kit nach Anspruch 1, dadurch gekennzeichnet, daß die Vertiefungen der Mikrotiterplatte zusätzlich ein Enzym- oder Biotin-gekoppeltes Konjugat eines Sekundärbindungspartners , insbesondere -antikörpers , und im Fall des Biotin-gekoppelten Konjugates ein Enzym in lyophilisierter Form enthalten.2. Immunoassay kit according to claim 1, characterized in that the wells of the microtiter plate additionally contain an enzyme or biotin-coupled conjugate of a secondary binding partner, in particular antibody, and in the case of the biotin-coupled conjugate contain an enzyme in lyophilized form.
3. Immun-Test-Kit nach Anspruch 1 oder 2 , dadurch gekennzeichnet, daß die Vertiefungen der Mikrotiterplatte ein Probenverdünnungsmedium in lyophilisierter Form enthalten.3. Immunoassay kit according to claim 1 or 2, characterized in that the wells of the microtiter plate contain a sample dilution medium in lyophilized form.
4. Immun-Test-Kit nach Anspruch 1 , 2 oder 3 , dadurch gekennzeichnet , daß der Test-Kit zusätzlich zur vorbeschichteten Mikrotiterplatte lediglich Waschpuffer, Substratlösung und Stopplösung enthält .4. Immunoassay kit according to claim 1, 2 or 3, characterized in that the test kit contains only wash buffer, substrate solution and stop solution in addition to the precoated microtiter plate.
5. Immun-Test-Kit nach einem der Ansprüche 1 bis 4 , dadurch gekennzeichnet, daß im Falle von Biotin-gekoppeltem Sekundär- antikörper-Konjugat als Sekundärreagenz Streptavidin - Horse Radish Peroxidase (HRP) eingesetzt ist .5. Immune test kit according to one of claims 1 to 4, characterized in that in the case of biotin-coupled secondary antibody conjugate is used as a secondary reagent streptavidin - Horse Radish Peroxidase (HRP).
6 . Immun-Test-Kit nach einem der Ansprüche 1 bis 5 , dadurch gekennzeichnet, dass der Test-Kit Stabilisatoren für Peroxidase beziehungsweise generelle Lyoprotektanten wie Proteine (z.B. Albumin (z.B. BSA) Protein Hydrolysate (Peptone, Gelatine...) Casein) und/oder Polymere (z.B. Dextran, PVA, PVP) , Zucker (z.B. Saccharose, Trehalose, Lactose, Xylit, Sorbit, Mannit, Maltose, Glucose, Inosit) und/oder Bakteriostatische -Mittel ~(z_B. „Thi erosal ,_ Proclin) und/ oder phenolische Substanzen und Aniline auch mit Substituenten (kleine Alkyl-Reste oder Cl, Br...) (z. B. o-Methoxyphenol, o -Methylphenol, p- Methylphenol , o-Aminophenol , o-Hydroxybenzoesaäure, (o, m oder p) -Hydroxybenzylalkohol, Anilin, p-Aminobenzoesäure, p-Methoxy- anilin, Benzylalkohol, Benzoesäure, p-Nitrophenol, Benzylamin, 1-Phenyl-l , 2-ethandiol, trans-1, 2-Cyclohexandiol, cis-1, 2-Cyclo- hexandi carbonsäure, Cyclohexylamin) und/oder Hydrophobe Verbindungen und/ oder Lösungsmittel (z.B. DMF, Ethylenglykol, DMSO) und/oder Detergenzien (z.B. Tween-20) und/oder Aryl-Borsäurever- bindungen (z.B. Phenylbor säure, -Brom- Phenylbor säure, 3-Aceta- midophenylborsäure, 1-Naphtylbor säure) und/ oder Substratanaloge (z.B. TMB, Luminol) und/oder Poly-Hydroxy-Verbindungen (z.B. Polyole, Polyethylenglykol, Glyzerin) und/oder Ξctoine (z.B. (S) -2-methyl-l ,4,5, 6-tetrahydropyrimidine-4-carboxylic-acid [THP(B) ] , (S,S)-ß-2-methyl-5-hydroxy-l, 2,4,5, 6-tetrahydropyri- midine-4-carboxylicacid, [THP(A)]) und/oder Ionen bzw. polyvalente Ionen (z. B. Metallionen (AI, Zn, Mg, Fe, Cu, ... ) ) und/oder Komplexbildner (z.B. EDTA) und/oder Aminosäuren (z.B. Glyzin, Prolin, 4-Hydroxyprolin, Serin, Glutamat, Alanin, Lysin, Sarcosin, y-Aminobutyruc acid, Phenylalanin) und/oder Substanzen wie TRIS, Salze, A ine, Na-Cholate, Saccharose onolaurat und/oder 2-O-ß-Mannosylglyzerat enthält.6. Immune test kit according to one of claims 1 to 5, characterized in that the test kit stabilizers for peroxidase or general lyoprotectants such as proteins (e.g. albumin (e.g. BSA) protein hydrolysates (peptones, gelatine ...) casein) and / or polymers (e.g. dextran, PVA, PVP), sugar (e.g. sucrose, trehalose, lactose, xylitol, sorbitol, mannitol, maltose, glucose , Inositol) and / or bacteriostatic agents ~ (e.g. "Thi erosal, _ proclin) and / or phenolic substances and anilines also with substituents (small alkyl residues 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-methoxy-aniline, benzyl alcohol, benzoic acid, p-nitrophenol, benzylamine, 1-phenyl-l, 2-ethanediol, trans-1, 2-cyclohexanediol, cis-1, 2-cyclohexanedicarboxylic acid, cyclohexylamine) and / or hydrophobic compounds and / or solvents (e.g. DMF, ethylene glycol, DMSO) and / or detergents (eg Tween-20) and / or aryl boric acid compounds (eg phenylboric acid, bromophenylboric acid, 3-acetamidophenylboric acid, 1-naphthylboric acid) and / or subs pedal analogs (e.g. TMB, luminol) and / or poly-hydroxy compounds (e.g. polyols, polyethylene glycol, glycerin) and / or octoins (e.g. (S) -2-methyl-l, 4,5, 6-tetrahydropyrimidine-4-carboxylic -acid [THP (B)], (S, S) -ß-2-methyl-5-hydroxy-l, 2,4,5, 6-tetrahydropyrimidine-4-carboxylic acid, [THP (A)]) and / or ions or polyvalent ions (e.g. B. metal ions (Al, Zn, Mg, Fe, Cu, ...)) and / or complexing agents (eg EDTA) and / or amino acids (eg glycine, proline, 4-hydroxyproline, serine, glutamate, alanine, lysine, sarcosine , y-Aminobutyruc acid, phenylalanine) and / or substances such as TRIS, salts, A ine, Na cholates, sucrose onolaurate and / or 2-O-ß-mannosylglycerate.
7. Verwendung eines Immun-Test-Kits nach einem Ansprüche 1 bis 6, für den Einsatz in der Forschungsdiagnostik und in-vitro Diagnostik, für den Einsatz in der Erregerserologie, wie zum Beispiel zum Nachweis von Infektionen durch HIV, HepV , EBV, CMV usw. , für den Einsatz in der Tumordiagnostik, wie zum Beispiel zum Nachweis von Tumormarkern oder Tumor assoziierten Proteinen, Vaskularisierungsmarkern, Metastasierungsmarkern usw. , für den Einsatz in der Allergologie wie zum Beispiel zum Nachweis von Tierall er genen, Pflanzenpollen, Nahrungsbestandteilen usw. , für den Einsatz in der Autoimmun-Diagnostik wie zum Beispiel zum Nachweis der Auto-Antigene im Bereich ANA/ENA, Diabetes mellitus/ IDDM, Schilddrüsen Antigene, Autoimmun Hepatitis , APS , usw . , für den Nachweis von immunologischen Störungen durch -entzündliche -Prozesse- oder- -Infektionen -wie— zum -Beispiel- -zum - Nachweis von Zytokinen, Adhäsionsmolekülen, Chemokinen, usw. , für den Nachweis von Änderungen des Hormonhaushaltes wie zum Beispiel Ovulationstests , Schwangerschaftstests , usw. , für den Einsatz zum Therapie-Monitoring wie zum Beispiel zum Nachweis von therapeutischen Antikörpern und Antigenen, organischen und anorganischen Verbindungen, für den Nachweis von Zelltod durch Apoptose oder Nekrose und/oder für den Nachweis von genetischen Veränderungen, genetischen Erkrankungen . 7. Use of an immunoassay kit according to one of claims 1 to 6, 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, vascularization markers, metastatic markers etc., for use in allergology such as for example for the detection of animal allergens, plant pollen, food components etc., For use in autoimmune diagnostics such as for the detection of autoantigens in the area of ANA / ENA, diabetes mellitus / IDDM, thyroid antigens, autoimmune hepatitis, APS, etc. , for the detection of immunological disorders caused by inflammatory processes or infections, such as for example for the detection of cytokines, adhesion molecules, chemokines, etc., for the detection of changes in the hormone balance such as ovulation tests, Pregnancy tests, etc., for use in 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.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AT0037001U AT5044U1 (en) | 2001-05-10 | 2001-05-10 | QUANTITATIVE ONE-STEP IMMUNITY TEST IN LYOPHILIZED FORM |
| AT3702001U | 2001-05-10 | ||
| PCT/AT2002/000112 WO2002090983A2 (en) | 2001-05-10 | 2002-04-26 | Quantitative single-step immunoassay in lyophilised form |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP1388012A2 true EP1388012A2 (en) | 2004-02-11 |
Family
ID=3488777
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP02769103A Withdrawn EP1388012A2 (en) | 2001-05-10 | 2002-04-26 | Quantitative single-step immunoassay in lyophilised form |
Country Status (12)
| Country | Link |
|---|---|
| US (1) | US20040171087A1 (en) |
| EP (1) | EP1388012A2 (en) |
| JP (1) | JP2004527758A (en) |
| CN (1) | CN1507565A (en) |
| AT (1) | AT5044U1 (en) |
| CA (1) | CA2446345A1 (en) |
| CZ (1) | CZ20033209A3 (en) |
| HU (1) | HUP0400081A3 (en) |
| IL (1) | IL158393A0 (en) |
| PL (1) | PL366665A1 (en) |
| SK (1) | SK13012003A3 (en) |
| WO (1) | WO2002090983A2 (en) |
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| ITMI20061063A1 (en) * | 2006-05-31 | 2007-12-01 | Mindseeds Lab S R L | METRODO AND PE SYSTEM RLA SELECTION AND MODIFICATION OF SINGLE CELLS AND THEIR SMALL AGGREGATES |
| KR100900985B1 (en) | 2007-08-28 | 2009-06-04 | 한국기계연구원 | Method for fixation of micro structure inside using?lyophilization |
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| CN114324882B (en) * | 2020-10-12 | 2022-12-27 | 广东菲鹏生物有限公司 | Protein stabilizer and application thereof |
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- 2002-04-26 CN CNA028095553A patent/CN1507565A/en active Pending
- 2002-04-26 EP EP02769103A patent/EP1388012A2/en not_active Withdrawn
- 2002-04-26 CZ CZ20033209A patent/CZ20033209A3/en unknown
- 2002-04-26 US US10/476,993 patent/US20040171087A1/en not_active Abandoned
- 2002-04-26 PL PL02366665A patent/PL366665A1/en not_active Application Discontinuation
- 2002-04-26 WO PCT/AT2002/000112 patent/WO2002090983A2/en not_active Application Discontinuation
- 2002-04-26 HU HU0400081A patent/HUP0400081A3/en unknown
- 2002-04-26 SK SK1301-2003A patent/SK13012003A3/en unknown
- 2002-04-26 CA CA002446345A patent/CA2446345A1/en not_active Abandoned
- 2002-04-26 IL IL15839302A patent/IL158393A0/en unknown
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Also Published As
| Publication number | Publication date |
|---|---|
| CA2446345A1 (en) | 2002-11-14 |
| SK13012003A3 (en) | 2004-04-06 |
| JP2004527758A (en) | 2004-09-09 |
| CZ20033209A3 (en) | 2004-04-14 |
| HUP0400081A3 (en) | 2004-05-28 |
| PL366665A1 (en) | 2005-02-07 |
| US20040171087A1 (en) | 2004-09-02 |
| WO2002090983A2 (en) | 2002-11-14 |
| CN1507565A (en) | 2004-06-23 |
| AT5044U1 (en) | 2002-02-25 |
| IL158393A0 (en) | 2004-05-12 |
| WO2002090983A3 (en) | 2003-09-12 |
| HUP0400081A2 (en) | 2004-04-28 |
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