EP0483309A1 - Solid support having antibodies immobilized thereon - Google Patents

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
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
• • 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
  • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
  • G01N33/552—Glass or silica
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6854—Immunoglobulins
  • G01N33/6857—Antibody fragments

Definitions

• This invention relates to solid supports having antibodies immobilized thereon and, more particularly, to such solid supports which are useful in solid phase immunoassays.
• assay elements are known for use in immunoassays to analyze for analytes present in biological fluids.
• one member of the antigen-antibody interaction pair e.g., the antibody
• a solid carrier such as particulate materials, fibrous materials and the like.
• the solid support becomes the site at which the interaction between the bound material and a binding partner takes place.
• any of the known immunometric assays can be carried out in the solid phase format.
• Such assays are well known to those skilled in the art. Briefly, for example, in the competitive assay a sample fluid containing an analyte of interest, e.g., an antigen, is applied to a solid support having bound thereto a binding partner of the analyte, e.g., an antibody. The amount of antibody bound to the support is that necessary to allow the requisite competition between the sample analyte and the labeled species.
• a labeled analyte or a labeled analogue thereof is added to the solid support and after another incubation period and a wash step to remove any unbound labeled material, the bound labeled material is detected to provide a signal which is a function of the amount of analyte in the patient sample fluid.
• the signal is inversely proportional to the amount of analyte in the sample.
• the antibodies which are immobilized on the solid carrier can be bound thereto by various means including covalent binding, immunoprecipitation and the like.
• the immunoprecipitation technique can be carried out more conveniently as compared to the chemical reaction(s) required for covalent binding.
• assays such as the competitive immunoassay described previously, only a relatively limited amount of antibodies may be bound to the carrier because of various factors such as the assay concentration range, otc. If the binding technique employed is not efficient enough a low signal level may be obtained with a given amount of antibodies. Of course, the signal can be improved by adding more antibodies to the solid carrier but this alternative is not satisfactory if it results in the assay range being shifted outside of the range required for the analyte of interest.
• the mouse monoclonal anti-digoxin antibodies are included in a complex together with an anti-mouse antibody which is raised against the Fc fragments of mouse antibodies and a mouse IgG antibody which is nonspecific.
• the complex is immobilized on the solid support by passive adsorption.
• the solid supports of the invention are prepared by initially mixing the antibody of interest, e.g., the anti-digoxin antibody, with the nonspecific antibody in a coating solution, typically a buffer solution, and then adding the specific antibody raised against the Fc fragments of the specific and nonspecific antibodies. The mixture is incubated under time and temperature conditions which allow the complex to form. The coating solution is applied to the solid support and the latter is dried.
• the antibody complex is immobilized to the solid support through passive adsorption.
• the solid supports of the invention have been found to be particularly advantageous when used in competitive solid phase immunoassays for the analysis of analytes in a sample fluid. Higher signal levels can be obtained while at the same time maintaining the sensitivity of the assay in the desired assay range.
• the solid supports of the invention may comprise any suitable solid carrier such as, for example, particulate materials, porous members possessing an intercommunicating network of openings throughout such that a fluid deposited on the member will propagate through the member, and the like.
• suitable porous members include porous membranes, fibrous mesh materials and the like and may be of any suitable material such as glass, polymeric materials, paper, etc.
• the solid carrier is a thin fibrous mesh member which will permit fluid deposited thereon to propagate throughout because of capillary action.
• the member may be any suitable fibrous material; a preferred material is a nonwoven glass fiber mesh, the fibers being very thin such as on the order of about 1 micrometer ( ⁇ m).
• any monoclonal antibodies of interest may be immobilized on the solid carrier such as, for example, antibodies to: haptens such as digoxin, triiodothyronine (T3) and the like; proteins such as myoglobin; hormones; etc.
• the nonspecific antibody may be any to which the antibody directed against the Fc fragments of the specific and nonspecific antibodies will bind.
• the nonspecific antibody is of the same species as the specific antibody, i.e., they are both raised in the same type of host animal, e.g., mice.
• the antibody which is raised against the Fc fragments of the specific and nonspecific antibodies can be raised in any host animal.
• the coating solution which is used to apply the antibody complex to the solid support is prepared by initially combining the specific antibody of interest and the nonspecific antibody in an appropriate solution, preferably a buffer solution.
• the solution may contain other additives such as, for example, proteins which are useful for reducing nonspecific binding, stabilizers such as sugars, surfactants which improve the wettability of the solid support and preservatives for preventing anti-microbial action.
• the antibody directed against the Fc fragments of the specific and nonspecific antibodies is added.
• the solution is then incubated under time and temperature conditions which are required to allow the complex formation to occur. These conditions will vary depending upon the specific materials, the amounts involved, etc. In a preferred embodiment the coating solution is incubated at 37°C for about one hour.
• the solution is then deposited on the solid support and allowed to become immobilized thereon through passive adsorption.
• the solid support is dried to provide a member which is useful in solid phase immunoassays.
• the solid supports of the invention are utilized in the known competitive solid phase immunometric assays for an analyte of interest. Such assays are well known to those skilled in the art and extensive discussion thereof is not required here. To further describe the advantages of the solid supports of the invention their use in a preferred fibrous solid phase competitive immunoassay will be described.
• an assay element 10 which includes a plurality of chambers in a housing 12 wherein a first chamber 14 serves as a front reservoir for the storage of a fluid reagent to be utilized in the assay, a second of the chambers serves as a back reservoir 16 for the storage of another fluid reagent for the assay, a third chamber 18 serves a mixing well for mixing reagents and fourth chamber 20 is adapted to dispense a fluid to one end of a porous solid support 22.
• a chamber 24 within the housing 12 wherein there is arranged an absorbing material for absorbing fluid removed from solid support 22 such as by a wash fluid as the latter propagates through the fibrous solid support.
• a frangible as puncturable foil (not shown) is disposed over the front and back reservoirs, 14 and 16, respectively, for containing the fluid reagents within these reservoirs thus providing a self contained assay element.
• the solid support 22 in this preferred embodiment comprises a thin fibrous mesh pad to which there has been immobilized an antibody of interest in accordance with the invention.
• a preferred solid support is a nonwoven glass fiber mesh having very thin fibers such as on the order of about 1 micrometer ( ⁇ m). This preferred solid support is from about 0.30 mm to about 0.60 mm in thickness, preferably about 0.40 mm.
• the solid support 22 extends from the dispensing chamber 20 to the chamber 24 which holds the absorbing material.
• the fluid absorbing material 26 which may be any suitable material, is located within chamber 24 and forms a part of the chamber 24 for taking up fluid expelled from the solid support and the guide area (not shown) in which the support is arranged.
• the absorbing material 26 is located contiguous the solid support 22 and in a preferred embodiment, as illustrated, is formed conveniently as an extension of the solid support material folded back and forth on itself.
• the housing 12 also includes a chamber 28 which is positioned immediately above the area of the solid support where the immunometric interactions take place. It will be apparent to those skilled in the art that the immobilized antibody complex bound to the solid support need only be present on that part of the support where the immunometric interactions will take place.
• the housing 12 also preferably includes a transparent window (not shown) or an opening in the housing, positioned immediately below the bottom surface of the solid support 22 to provide access for the readout illumination used to measure the detectable change effected in the support as a result of the assay method.
• a volume of sample fluid typically 30-45 ⁇ is deposited on the solid support 22 through chamber 28.
• the sample fluid is allowed to incubate for a suitable period at the appropriate temperature to allow the sample antigen to interact with the immobilized antibodies in the reaction site on the solid support.
• a conjugate of an enzyme-labeled antigen (the same as the sample antigen) or an analogue thereof is withdrawn from chamber 14 and deposited on the solid support through chamber 28.
• the conjugate will bind to any specific antibodies on the solid support which are not bound to the sample antigen.
• the assay element 10 is again allowed to incubate. Since the enzyme label must be detected indirectly, a substrate solution, typically 50-100 ⁇ is applied to the porous solid support 22 through chamber 20. The substrate solution is utilized both as a wash fluid to remove from the solid support any unbound sample antigen or conjugate and to render the enzyme label detectable.
• the substrate solution is caused to enter an end portion of the solid support and as the solution propagates through the solid support 22 it forces any unbound sample antigen and enzyme conjugate together with the fluid out of the solid support and into the absorber chamber 24 where it is taken up by the absorber material 26.
• the signal provided by the fluorescent species liberated by the reaction of the enzyme with the substrate material is read out by means of suitable optical apparatus.
• EXAMPLE I An antibody solution was prepared containing 0.1 g/ml of anti-digoxin antibodies and 10 ⁇ g/ml of mouse IgG antibodies in a pH 7.2 phosphate buffered saline solution which also contained other proteins, stabilizers, surfactants and anti-microbial agents. The solution was filtered through a 0.2 ⁇ m filter and then 2 ml of a solution of Goat Anti-Mouse IgG, Fc Fragment Specific (Jackson ImmunoResearch) was diluted 1 : 100 with the antibody solution to form a coating solution.
• the coating solution was incubated for one hour at 37°C and about 35 ⁇ added to an approximately 7 x 8 mm area of a Whatman GF/F fibrous glass filter material.
• the filter material was dried for about 10 minutes at 75 °C and incorporated into an assay element as described previously.
• the assay elements were then used to carry out the competitive immunometric assay method previously described with sample fluids containing known digoxin concentrations.
• the assays were carried out on an automated laboratory instrument.
• the enzyme label used in the conjugate was methyl umbelliferyl phosphate and the readout signal was obtained by directed 360 nm radiation upon the bottom surface of the solid support and measuring the reflected 450 nm radiation.
• the signals obtained are shown in Table I.
• the assays were read kinetically and the signals obtained are the slopes of the kinetic measurements. Each value shown in Table I is the average of three replicates.
• the assay method of the invention provided high signal levels and a desirable steep standard curve slope in the assay range (about 0.5 - 4.0 ng/ml).
• EXAMPLE II A coating solution was prepared which was the same as that described in Example I except that it contained 0.0625 / g/ml of a monoclonal antibody to T3 instead of the anti-digoxin antibodies. Assay elements were prepared as described in Example I and used to carry out the competitive solid phase assay method previously described. The results obtained are shown in Table II. Table II
• the assay method of the invention provided high signal levels and a desirable steep standard curve slope in the assay range (about 0.50 - 8.00 ng/ml).

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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Immunology (AREA)
• Engineering & Computer Science (AREA)
• Chemical & Material Sciences (AREA)
• Molecular Biology (AREA)
• Biomedical Technology (AREA)
• Urology & Nephrology (AREA)
• Hematology (AREA)
• Biotechnology (AREA)
• Analytical Chemistry (AREA)
• Cell Biology (AREA)
• Pathology (AREA)
• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Physics & Mathematics (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Inorganic Chemistry (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Description

Claims

Applications Claiming Priority (2)

Publications (1)

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• 1991
  • 1991-03-27 EP EP19910908038 patent/EP0483309A1/en not_active Withdrawn
  • 1991-03-27 WO PCT/US1991/002092 patent/WO1991018291A1/en not_active Application Discontinuation
  • 1991-03-27 JP JP50784291A patent/JPH05500116A/en active Pending
  • 1991-04-02 CA CA 2039599 patent/CA2039599A1/en not_active Abandoned

Non-Patent Citations (1)

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