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
  • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
  • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
  • G01N33/538—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by sorbent column, particles or resin strip, i.e. sorbent materials
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing

Definitions

• the present invention relates to reaction columns for measure ⁇ ment via affinity assay with at least one substrate bed.
• Affinity chromatography is a technology which is broadly ap ⁇ plied in preparative purification of bio-molecules. Therein, advantage is taken of a specific interaction between the mole ⁇ cule to be determined and a complementary binding partner.
• a sample containing the bio-molecule to be purified is applied to a chromatographic column, which contains one of the mutually complementary bin ⁇ ding partners bound to a solid substrate.
• pairs of complementary binding partners are enzymes and their substra ⁇ tes, antibodies and antigens or haptens and mutually comple ⁇ mentary DNA or RNA single chains.
• HTLV-1 Human T-Leukemia Virus Type 1
• HIV-1 and HIV-2 Human Immunodeficiencies Virus 1 and 2
• HCV Hepati ⁇ tis C Virus
• the first group describes modified ELISA plate test which on one side can be used from multiple measurements but which have on the other side several draw backs.
• the other group are test methods which are based on column chromatogra ⁇ phy. Such methods are only available for single analysis me ⁇ thod and not for multi-measurements.
• the WO-A-91/13354 describes a flow immunosensor method and an apparatus for performing this method.
• the method of detecting a target moiety comprises the steps of providing an antibody specific to the target, saturating the antibodies binding sites with a labelled form of the target, flowing a liquid containing the target past the antibody, allowing the target to displace the labelled antigen and detecting the displaced, labelled antigen.
• the EP-A-0473 065 discloses an assay to simultaneously detect the presence of one or more analytes in a test sample.
• the analytes are captured on different solid phases and the pre ⁇ sence of one or more analyte is determined by detecting a signal generated by analyte-specific indicator reagents.
• This assay is a modified ELISA plate test which needs long incuba ⁇ tion times and is difficult in temperature control.
• WO-A-92/12255 describes assays for the determination of mul ⁇ tiple analytes which may be present in a test sample by using different short-lived and long-lived chemiluminescent labels and integrating the generated chemiluminescence signal and time discriminating the short-lived and the long-lived com ⁇ ponents of the signal generated. Also provided are assay kits which contain these short-lived and long-lived chemilumines ⁇ cent compounds. This method also describes an ELISA plate test with long incubation time and difficult temperature control.
• the DE-A-42 08 732 and the DE-C-41 26 436 of the applicant describe reaction columns for solid phase immuno- analysis and a method to determine components detectable by immunoreactions. These columns are easy to handle, work in flow through procedure, need short reaction times and give quantitative reactions. After a basic standardization of the respective lot of columns, further prior calibration or rege ⁇ neration are not necessary nor a reference standard sample measurement.
• reaction column for simultaneous multiple measurement which allows the rapid performance of simultaneous qualitative or quantitative analysis of several components or parameters with solid phase affinity analysis and has the same advantages as the columns described above.
• the object of the present invention is solved by a reaction column for simultaneous multiple measurement via affinity assay with at least one substrate bed comprising at least one affinity reactive component being bound to each substrate bed and the volume of each bed being up to 600 ⁇ l.
• the substrate bed is a solid porous material or a gel.
• the substrate bed is positioned between two porous separating devices.
• the reaction column can contain components for compound-specific affinity reactions or components for group-specific affinity reactions or both. These compounds can be immunologically reactive.
• the reaction column according to the invention is prepared with a substrate bed and a reactive component for the affinity reaction in confected and standardized form, allowing imme ⁇ diate use of the column without any calibration steps or refe ⁇ rence measurements.
• the confection has to happen after the quantitative reaction.
• the reactive components for group-specific affinity reactions and for compound specific affinity reactions have preferably such a loading density that the compounds are bound quantita ⁇ tively to the substrate bed during the controlled flow of the liquid sample before it leaves the column.
• the quantitatively bound compounds are the compounds to be determined, or a group containing them or affinity reactive secondary binding partners for the compounds which are applied to the column in any sensible order and number of steps.
• the reactive components on the sub ⁇ strate bed are protein A, protein B, protein G, protein M, an immunoglobulin or a group of immunoglobulins, an antigen or a group of antigens.
• binding pairs can include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors and enzymes, and the like.
• specific binding pairs can include members that are analogs of the original specific binding member, for exam ⁇ ple, an analyte-analog.
• Immunoreactive specific binding mem ⁇ bers include further antigens, antigen fragments; antibodies and antibody fragments, monoclonal and polyclonal; and com ⁇ plexes thereof, including those formed by recombinant DNA me ⁇ thods. Also in the antigen and the group of antigens hapten is included.
• the substrate material can be selected from the group contai ⁇ ning polymeric sugars, plastics, modified plastic organic or inorganic support materials, porous metals, metal oxides, and alloys, glasses, silicates, or ceramics.
• polymeric sugar agarose can be used as polymeric sugar agarose.
• the reactive component can be bound to the substrate by cova- lent or any kind of sorptive binding.
• porous frits or membranes can be used, if neces ⁇ sary.
• the frits have a porosity of 0.2 ⁇ m to 100 ⁇ m and are made of plastic material, e.g. polyethylene, metal or glass.
• plastic material polyethylene or teflon can be used and as metal porous aluminum or stainless steel.
• the column material further can be selected from the group containing plastics, metals, and natural materials, for example, polyethylene, polypropylene and/or polystyrene as plastic material.
• reaction columns can be shaped to allow male-female connections for in series connection of se ⁇ veral reaction columns.
• the substrate bed has preferably a volume of 30 to 50 ⁇ l.
• sequence of substrate bed can be arranged in such a manner that a compound which is disturbing the affinity reaction of another compound, is bound quantita ⁇ tively in an upper substrate bed, while the affinity reaction of the other compound takes place in a lower substrate bed.
• additional beds or zones can be in ⁇ tegrated above or below the measuring substrate beds, such as filtration devices or zones for sample purification or treat ⁇ ment, additional reaction zones for preparation of the affini ⁇ ty binding reaction, or control zones for control of sample application, sample flow, and of applied chemicals.
• the invention further relates to a method for the determina ⁇ tion of compounds being determinable via an affinity reaction, wherein a sample to be analyzed is applied to a reaction co ⁇ lumn described above, and the compounds to be analyzed or a group containing them are bound quantitatively by the comple ⁇ mentary reactive component in the reaction column, whereby the components to be analyzed are determined thereafter by known determination methods after known affinity assay procedures in the reaction column.
• An affinity reaction may be used before to bind the complemen ⁇ tary reactive component itself to the column, loaded with a complementary reactive component to this complementary reac ⁇ tive component (see example 3) . This allows more flexible use of the column, i.e. for different assays and for assay deve ⁇ lopment, including hiding of the real application of the final user to the producer of the column.
• Such methods can be secondary affinity reaction in the reac ⁇ tion column for labelling and/or amplification and determi ⁇ nation in the column, or determination after elution.
• affinity reaction of labelled compounds competing with compounds to be determined and determination directly in the liquid leaving the column, directly in the column, or after elution is possible.
• the group bound by the group-speci ⁇ fic component is the group of immunoglobulines G and/or M.
• a solution of similarly labelled antigens with one enzyme or fluorescence label for all com ⁇ plementary antigens can be applied for the qualitative deter ⁇ mination of the presence of any of special immunoglobulines G and/or M.
• a solution of differently labelled antigens can be applied and the anti ⁇ genes are bound via affinity reactions in direct relation to the amount of bound corresponding immunoglobulines G and/or M.
• the different antigen labels are different enzyme or fluores ⁇ cence labels for each complementary antigen. Furthermore, control affinity reactions in respective substra ⁇ te beds can be used to control the application and the flow of the sample liquid and the quality of the applied reagents.
• reaction column can directly be connected to and remaining at the container of the liquid to be tested, or a blood container especially directly to its filling device for the purpose of safe sampling and test ap ⁇ pointment. The test result can thus be seen in the moment of usage.
• the method can be performed without calibration and without regeneration of the substrate bed and of the comple ⁇ mentary reactive component. Additionally, small suction or pressure forces can be applied to the reaction column after sample application for more rapid performance. Further a han ⁇ dling automate can be used for automated sampling, sample ap ⁇ plication, and/or measurement.
• reaction column and the method of the invention it is possible to determine qualitatively or quantitatively, for example, all or specific immunoglobulines of the classes IgG, IgM, IgA and/or IgE in body fluids.
• the presented method using the reaction column according to the invention is much simpler than common procedure and allows a rapid quantitative and qualitative determination. This is enabled partly because the determination of the component to be determined does not require a preceding calibration or regeneration of the used substrate in the column and because no series reference measurements of standard solutions are necessary.
• the present procedure can be performed rapidly under the force of gravitation without application of high pressure to the reaction column after sample application. But low suction or pressure forces can be used for quicker performance, for exam ⁇ ple, by centrifugation or pumping. This may be important for substrate beds with large flow resistance to keep the time for flow-through and thus the test time low.
• the sample to be analyzed is applied to a reaction column with several different immunolo- gically reactive components .
• the components to be determined are retained by the complementary immunologically reactive component (s) .
• the compounds are quantitatively or qualitatively analyzed as described in DE-C-41 26 436 via fluorescence and/or an enzyme colour reaction in the substrate bed in the liquid leaving the column after solution applica ⁇ tion, or after elution.
• fluorescent and/or an enzyme colour reaction in the substrate bed in the liquid leaving the column after solution applica ⁇ tion, or after elution.
• different labels can be used at the antigen or antibody or further com ⁇ plementary binding partners.
• the concentration of the compounds to be analyzed is very low, sample enrichment can be performed by applying larger amounts of sample liquid, if available.
• the reaction co ⁇ lumn is loaded with an antibody or a group of antibodies com ⁇ plementary to the respective class or classes of antibodies (e.g. to human IgG and/or IgM and/or IgA) or a respective group-specific reactive component or respective group-specific reactive components such as protein A or G.
• a solution with an antigen of the component or with antigens of the components to be analyzed is applied to the column.
• This method has the advantage, that it has a higher sensibili ⁇ ty, can be better adjusted and that one type of column can be used for different multi- or single tests.
• the specific kind of the test is determined by the antigens applied afterwards to the column. This is more effective from economical reasons.
• several separated substrate bed volumina with diffe ⁇ rent immunological and other reactive components can be used in one or more reaction vessel one upon another.
• dif ⁇ ferent separating devices are used in the column, for example, membranes or frits or several substrate beds with different materials which are positioned between porous separating devi ⁇ ces.
• a reac ⁇ tion column For a sample with several components to be analyzed, a reac ⁇ tion column is used with the number of separated substrate beds corresponding to the number of components. These substra ⁇ te beds are loaded with one or several complementary reactive partners for the compounds or group of compounds to be ana ⁇ lyzed. During the sample flow the corresponding compound is retained in the respective substrate bed and, for example, indicated by colour reaction. Disturbing similar compounds with high affinity binding constants are removed quantitati ⁇ vely and are determined, if wanted, in the upper substrate beds. In the lower bed(s), the determination of the com ⁇ pound(s) with lower affinity constant (s) follow(s) .
• the reactions are performed in dif ⁇ ferent suitable gel beds within one column. Also a simulta ⁇ neous test of small and large components (antibodies/antigens, bacteria or viruses) is possible, if an upper gel bed is as ⁇ signed for the larger components, as described in DE-A- 42 08 732.
• control fields are introduced in the upper and lower part of the co ⁇ lumn.
• the upper negative control proves the exact flow of the sample
• the lower positive control proves, if the reactive com ⁇ pounds were loaded on the column in the right way, and if the sample applied to the column and the used reagents are in good quality.
• the reaction column After having performed the test, the reaction column remains at the tested sample, which should be interesting for blood banks.
• the test column can be connected before the test with a blood container, for example, with a T-piece between the container and the vene of the blood giver. Then the test is performed after taking of the blood sample with the remaining blood in the connected tube. The test vessel remains at the blood container, so that the test result is recognizable at any time without any documentation error.
• Figure 1 shows the reaction column according to the invention.
• the column has n substrate beds and is used for simultaneous multiple measurement.
• 1,2,3, ... ,n represent the different sub ⁇ strate beds with the attached reaction compounds.
• 11,12,13,14, 15,...,n' are seperating devices.
• Figure 2 shows the standard curve for the simultaneous detec ⁇ tion of tetanus and diphtheria antibody titers (see table 2 and example 2) .
• Figure 3 shows the standard curve for antibodies in slaughter cattle (see table 3 and example 3) .
• the an ⁇ tibody binding capacities for 30 ⁇ l sepharose beds were about 20 ⁇ g for antibody-loaded resp. 200 ⁇ g for protein G-loaded substrate beds.
• the beds were closed carefully with upper second frits 12. In the case of multi-field columns, this first procedure gave the respective lowest substrate bed 1, and was then carefully repeated for each of the further neces ⁇ sary substrate beds above the first one (beds 2-n, closing frits 13-n' ) .
• the columns were closed at the lower tips by caps, about 100 ⁇ l washing buffer was filled into each column above the upper frit, and the upper ends were closed by caps as well.
• the fluorescence detector Kontron SFM 25 was calibrated with a normalization solution, bovine serum albumin in PBS buffer (0.01 M Na 2 HPO 4 -0.01 M NaH 2 PO 4 -0.15 M NaCl-0.005 % Tween 20-pH 7.2), stabilized with 0.1 % sodium azide.
• Reagents - Washing buffer (0.01 M Tris-0.15 M NaCl-0.05 % Tween 20-pH 8.0)
• Tetanus vaccinations are performed in the case of injuries for prophylaxis, mostly. At (already or still) existing sufficient vaccine protection of the patient, the vaccination may cause anaphylactic shock even with fatal outcome.
• the determination of the vaccination status may as well be important before the application of the combined vaccines for tetanus and diphthe ⁇ ria (the latter is increasing again in Eastern Europe), i.e. for the decision whether to apply one single vaccine only instead of both.
• Reagents - Washing buffer (0.01 M Tris-0.15 M NaCl-0.05 % Tween 20-pH 8.0)
• Reagents - Washing buffer (0.01 M Tris-0.15 M NaCl-0.05 % Tween 20-pH 8.0)
• the fluores ⁇ cence was determined at 490/520 resp. 555/585 resp. 650/680 nm. The measurements were repeated twice one and two days later, and the same normalized standard line was used (see table 3 and figure 3) .
• the normalized fluorescence can also just be used for the qualitative evaluation, i.e. whether the sample contains forbidden antibiotics of this group.
• Rapid qualitative screening was also done using enzyme label ⁇ ling.
• the procedure was the same as in a) up to the last wa ⁇ shing step before the elution, but using a mixed solution of similarly phosphatase-labelled instead of differently fluores ⁇ cence-labelled antigens.
• Substrate buffer was then added, followed by an incubation time of 10 minutes. Evaluation was done by comparison of the columns with applied samples to a control column with an applied negative sample. The columns with positive samples all were only slightly yellowish, com ⁇ pared to the significantly dark yellow colour of columns with negative samples and of the control column.
• Rhodamin RFUS 453
• Rhodamin RFUS 489
• Rhodamin RFUS 480
• NFU RFU /RFUS of Normalisation Solution
• Reagents - Washing buffer (0.01 M Tris-0.15 M NaCl-0.05 % Tween 20-pH 8.0)
• 500 ⁇ l solution 250 ⁇ l human serum, diluted with 250 ⁇ l wa ⁇ shing buffer
• 300 ⁇ l of the mixed secondary antibody solution were applied, and the columns were incubated for about 6 minutes until the upper negative control field 5 became slightly yellowish (incubation time for enzyme reaction, not for affinity reaction) .
• test fields 2-4 became slightly yellowish only. All positive samples caused a dark yellow colour in at least one antigen test field.

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• 1994
  • 1994-01-13 JP JP7518787A patent/JPH09507577A/ja not_active Ceased
  • 1994-01-13 WO PCT/EP1994/000086 patent/WO1995019569A1/en not_active Application Discontinuation
  • 1994-01-13 AU AU58829/94A patent/AU5882994A/en not_active Abandoned
  • 1994-01-13 EP EP94905052A patent/EP0739487A1/en not_active Ceased
• 1995
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