EP0670040A1 - Immobilisation de proteines chimiquement reticulees sur des supports solides - Google Patents

Immobilisation de proteines chimiquement reticulees sur des supports solides

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Publication number
EP0670040A1
EP0670040A1 EP94928017A EP94928017A EP0670040A1 EP 0670040 A1 EP0670040 A1 EP 0670040A1 EP 94928017 A EP94928017 A EP 94928017A EP 94928017 A EP94928017 A EP 94928017A EP 0670040 A1 EP0670040 A1 EP 0670040A1
Authority
EP
European Patent Office
Prior art keywords
specific binding
solid phase
reagent
cross
linked
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94928017A
Other languages
German (de)
English (en)
Inventor
Spencer H. Lin
Pratap Singh
Steven E. Diamond
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Baxter Healthcare Corp
Original Assignee
Baxter Diagnostics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Baxter Diagnostics Inc filed Critical Baxter Diagnostics Inc
Publication of EP0670040A1 publication Critical patent/EP0670040A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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

  • This invention relates generally to the field of methods for immobilizing specific binding assay reagents on solid supports.
  • this invention relates to a. method of immobilizing cross-linked reagents on solid phase supports.
  • This invention also relates to a solid phase support having immobilized cross-linked reagents useful in diagnostic tests.
  • In vitrc diagnostic assays may be used to measure amounts of an analyte found in a body fluid sample or tissue sample.
  • the analyte must be distinguished from other components found in the sample.
  • Analytes may be distinguished from other sample components by reacting the analyte with a specific receptor for that analyte. Assays that utilize specific receptors to distinguish and quantify analytes are often called specific binding assays.
  • receptors are antibodies, fragments of antibodies and specific binding proteins such as Intrinsic Factor or Folate Binding Protein.
  • Receptors are characterized by having a reversible specific binding affinity for an analyte or an analog of that analyte.
  • the analog generally is an analyte derivative carrying a detectable marker such as an enzyme, fluorescent molecule or other known label which binds to a receptor with about the same specificity and affinity as the analyte.
  • the receptor or other assay reagent of the specific binding reaction is often immobilized on a solid phase.
  • Immobilization of these receptors is required to separate the bound components from the unbound components.
  • the various methods by which a receptor or other reagent is immobilized on a solid phase include adsorption, absorption and covalent bonding. Procedures have been described for immobilizing an essentially soluble immunocomplex of a receptor and an antiserum to the receptor on an inert glass fiber solid phase support. These procedures are disclosed in U.S. Patent No. 4,517,288, incorporated by reference herein.
  • soluble immunocomplexes are prepared by combining at least two immunochemically reactive substances with one another in solution. At least one such immunochemically reactive material is selected for its immunochemical specificity for an analyte of interest. This is defined as the primary antibody. For example, if the soluble immunocomplex is to be used in an immunoassay for the detection of thyroid stimulating hormone ("TSH”), then one component of the immunocomplex is selected for its immunochemical specificity for TSH).
  • TSH thyroid stimulating hormone
  • TSH TSH.
  • a typical example would be an antibody with specificity for TSH, i.e., an anti-TSH antibody.
  • the second component of the immunocomplex could comprise an antibody preparation directed against the anti-TSH antibody.
  • Antiserum to anti-analyte antibodies for example to mouse anti-TSH antibodies, can be prepared by injecting purified mouse antibody into a host animal (e.g., a goat), and thereafter harvesting the antiserum to the mouse antibody. The mouse anti-TSH antibody and the goat antiserum to mouse antibodies are thereafter worked up as standard stock solutions.
  • Radial partition immunoassay (“RPIA”) is a type of specific binding assay that typically utilizes an inert glass fiber solid phase support. The glass fiber support has low non-specific binding.
  • a portion of a stock solution of the primary antibody is combined with the stock solution of the second component in a buffered medium.
  • the resulting immunocomplex in an appropriate volume of buffer, is thereafter spotted onto a delimited area of the glass fiber filter.
  • the two components of the immunocomplex may be applied to the filter as separate buffered solutions and allowed to react in situ. In both instances, the point of application of the immunocomplex defines a reaction zone within the solid phase. The applied immunocomplexes become adsorbed and entrapped within the interstices of the beds of fibers within the glass fiber filter.
  • the method of application can include dispensing the immunocomplex solution with a manual or automated pipette, or with other automated equipment including assay analyzer instruments. Subsequent to applying the immunocomplex to the solid phase, after incubating the immunocomplex for a suitable incubation period, the solid phase is dried under controlled conditions. This process yields a stable reactive reagent which can be used in any one of a number of solid phase specific binding assay protocols.
  • the sample and other reactants are applied to the solid phase so that they react with the immobilized receptor in the reaction zone. Separation of free analyte and/or analyte analog from bound analyte and/or analog is accomplished by application of a wash solution, with resultant chromatographic separation of the bound and unbound reagents on the solid phase support. This results in the reaction zone being essentially free of unbound materials and allows the monitoring of the amount of labelled reagent in the reaction zone.
  • the soluble double immunocomplex is formed by mixing the primary antibody, either monoclonal or polyclonal, with secondary polyclonal antibody as described in U.S. Patent No.
  • a further shortcoming of traditional RPIAs is that antibody fragments cannot be effectively used in these assays.
  • a complete monomeric immunoglobulin molecule can be enzymatically digested by pepsin into an F(ab') 2 fragment, an Fc fragment and other subfragments.
  • the F(ab') 2 fragment contains the specific antigen-binding activity of the antibody.
  • the Fc fragment contains no antigen-binding capability but nevertheless determines important biologic characteristics of the intact molecule.
  • One possible reason why F(ab') 2 fragments have not been effectively used in radial partition immunoassays is that the F(ab') 2 fragments may not be of sufficient size to remain imbedded in the matrix. Alternatively, such antibody fragments may be ineffective in the assay because they lack the carbohydrate portion of the Fc region. As a result, no immunocomplex is expected to form when F(ab') 2 fragments are used.
  • cross-linking reagents are used to couple specific binding receptors such as antibodies, F(ab') 2 fragments or other receptor proteins to other identical or related molecules.
  • the cross-linking agent is preferably a bifunctional reagent, preferably but not limited to bis(sulfosuccinimidyl) suberate, glutaraldehyde, or 1,4 butanediol diglycidyl ether.
  • the specific binding receptor can be a complete antibody molecule, an operable fragment of an antibody or a non-immunological receptor protein such as folate binding protein or intrinsic factor.
  • the cross-linked receptor protein complex can be immobilized onto a finite area of a solid phase support, such as a glass fiber matrix or polystyrene plate.
  • a solid phase support such as a glass fiber matrix or polystyrene plate.
  • the present invention comprises the method of immobilizing cross-linked receptor protein complexes on solid surfaces.
  • the invention further comprises solid phase reagents having cross-linked protein complexes immobilized thereon, as well as specific binding assay methods utilizing such solid phase reagents.
  • Figure 1 depicts the Stratus® A, B and F rates as functions of increasing concentrations of BS 3 cross-linked D70 antibodies.
  • Figure 2 depicts the Stratus® TSH assay using glutaraldehyde cross-linked CA2 antibodies as compared to CA2 antibodies bound by a secondary goat-anti-mouse (GAM) antibody on Stratus®.
  • Figure 3 depicts the Stratus® TSH assay using glutaraldehyde cross-linked CA2 antibodies as compared to CA2 antibodies bound by a secondary goat-anti-mouse (GAM) antibody on Stratus® II.
  • GAM secondary goat-anti-mouse
  • Clin. Chem. 28:1894-98 (1982) and in U.S. Patent No. 4,517,288, is an assay procedure in which all steps are conducted directly on a solid phase.
  • antibodies or other reagents are immobilized on a small area of glass fiber filter paper.
  • Various calibrators containing known amounts of an analyte to be detected or various unknown samples potentially containing such analyte are then allowed to react with this immobilized receptor.
  • excess reagents are removed from the center area of filter paper by application of a wash fluid.
  • the wash fluid may contain the substrate for the enzyme, thus initiating the enzyme reaction simultaneously with the wash step.
  • the action of the enzyme on the substrate generates a fluorescent signal.
  • the enzyme activity in a part of the center area is then quantifiable by front-surface fluorometry.
  • the rate of fluorescence is directly or inversely proportional to the concentration of analyte in the sample.
  • the solid phase present a relatively "inert" surface. That is, the surface should be relatively nonreactive with biological materials, particularly with respect to' nondiscriminate adsorption of proteinaceous materials.
  • the physical form of the solid phase is such that the interstices or pores within the solid phase are sufficiently small so that the reaction fluids are retained and transported by capillary action.
  • the solid phase pores or interstices should not be so small so as to retain undesirable components that might give rise to false positive signals.
  • the solid phase is advantageously composed of a mat of compressed fibers, such as glass or synthetic fibers or relatively inert cellulosic materials.
  • the solid phase also may be constructed of other porous constituents such as sintered glass, ceramics and synthetic polymeric materials.
  • Glass fiber filter paper is the preferred solid support of the present invention because of its inert characteristic and because of its ability to adsorb the cross-linked complexes of this invention in quantities sufficient for quantitative evaluation of retention of assay reagents.
  • the chemically cross-linked protein complexes of this invention once adsorbed onto a suitable solid phase, can be used in a wide variety of analytical protocols for analysis of a variety of biological materials.
  • chemically cross-linked protein complexes may be useful for immunoassay of urine, blood or blood components such as serum or plasma.
  • immunoassays can be directed to the detection or quantitation of therapeutic agents, natural or synthetic steroids, hormones, enzymes, antibodies and other analytes of interest.
  • Therapeutic agents that can be analyzed in such protocols include without limitation digoxin, dilantin, phenobarbital, theophylline, gentamicin, quinidine, and the like.
  • Solid phases prepared in the foregoing manner can also be used in immunoassays for the detection of steroids such as, without limitation, cortisol, aldosterone, testosterone, progesterone, and estriol or serum protein such as ferritin. Hormone levels are also capable of determination through the use of solid phase complexes of the present invention.
  • thyroid hormones such as tetraiodo- and triiodo-thyronine and thyroid stimulating hormone (TSH); peptide hormones such as corticotropin, gastriri, angiotensin, and proangiotensin; and polypeptide hormones such as insulin, thyrotropin, levteotropin, somatotropin and human chorionic gonadotropic hormone (HCG).
  • TSH thyroid stimulating hormone
  • peptide hormones such as corticotropin, gastriri, angiotensin, and proangiotensin
  • polypeptide hormones such as insulin, thyrotropin, levteotropin, somatotropin and human chorionic gonadotropic hormone (HCG).
  • HCG human chorionic gonadotropic hormone
  • complexes of the present invention include assay of relatively small molecules involved in metabolism, i.e., folate, to assay of polypeptide antigens and antibodies associated with infectious disease, e.g., antigens and antibodies associated with HIV, hepatitis, CMV, syphilis, Lyme disease agents, and numerous other infectious agents.
  • infectious disease e.g., antigens and antibodies associated with HIV, hepatitis, CMV, syphilis, Lyme disease agents, and numerous other infectious agents.
  • the chemically cross-linked protein complex /solid phase preparations of the present invention are applicable to a variety of specific binding assay formats. For example, various direct-binding assays may be employed with these reagents.
  • receptors such as antibodies or binding proteins are chemically coupled to make a cross-linked protein complex and the complex is immobilized on the solid phase.
  • the immobilized chemically cross-linked protein complexes are contacted with a sample containing the analyte of interest. Following binding of the analyte by the immobilized complex, the solid phase is washed and then contacted with an indicator.
  • indicator in the context of this invention means a labeled conjugate.
  • the conjugate comprises an antibody, antibody fragment, binding protein or analyte depending on assay format, and the label is a fluorescent, enzymatic, colorimetric, radiometric or other labeling molecule that is associated either directly or indirectly with the conjugate.
  • the label may be comprised of an enzymatic compound that produces fluorescence upon contact with a substrate.
  • the extent to which the indicator is present on the solid support can be correlated with the amount of unknown analyte as disclosed, for example, in Tijssen, P., Laboratory Techniques in Biochemistry and Molecular Biology. Practice and Theory of Enzyme Immunoassay, pp. 173-219 (Chapter 10) and pp. 329-384 (Chapter 14), Elsevier Science Publishers, Amsterdam, The Netherlands, 1985.
  • the complexes of the present invention also may be used in competitive assay formats.
  • the solid phase containing immobilized chemically cross-linked protein complexes with specificity for a selected analyte is contacted with a sample presumably containing such analyte and with a specific competitive reagent.
  • the specific competitive reagent may be a labeled analog of the analyte.
  • the labeled analog competes with the sample analyte for binding to a receptor immobilized on the solid phase.
  • analyte may be coupled to a solid phase and contacted with a sample and with a specific competitive cross-linked protein reagent, for example a labeled receptor for the analyte.
  • sample analyte competes with solid phase analyte for binding with soluble labelled cross-linked receptor.
  • the amount of label bound to the solid phase after washing provides an indication of the levels of analyte in the sample. That is, the amount of label bound to the soluble phase is inversely proportional to the amount of analyte in the sample.
  • Various instruments are available for applying the chemically cross-linked protein conjugates and various other binding assay reagents to a solid phase, washing, and reading the amounts of indicator bound to the solid phase.
  • the solid phase comprises glass fiber filter tabs that are analyzed using the Stratus®
  • the instrument is a batch-processing bench-top instrument, described by Giegel et al., Clin. Chem. 28:1894-98 (1982), incorporated by reference herein.
  • the instrument is adapted to process filter tabs in the radial partition immunoassay format, which format is also described in Giegel et al.
  • the instrument includes fluid dispensers for sample, conjugate and substrate washes. Microprocessor-controlled stepping motors aspirate and dispense required aliquots of reagents. All timing and operational aspects of the dispensers are predetermined by a program outine within the analyzer.
  • the instrument also includes a tab transport system, heated plate with temperature monitoring, sample and reagent fluid pumps, a read station, data processing, and means for tab disposal.
  • the instrument microprocessor control program periodically verifies critical operating conditions such as reference voltages, temperatures, and dispensing settings, and flags for out-of-limit values.
  • Solid phase supports used in the present experiments comprised "tabs" as used with the Stratus® analyzer instrument or the
  • the Stratus II® instrument is identical to the Stratus® instrument except that for analysis on Stratus®, all dilutions and /or mixing of the patient sample must be carried out off-line, whereas Stratus II® has the flexibility of programming for on-line dilutions of the patient samples with one or more solutions, prior to application to the solid phase.
  • the tabs are assembled from 1-in. (2.5 cm)-wide rolls of GF/F glass filter paper (Whatman Inc.) and snap-fit plastic tab parts, as disclosed in Giegel et al., Radial Partition Immunoassay, Clin. Chem. 28: 1894-98 (1982). Appropriate concentrations of cross-linked reagent solutions or control solutions are made up in spotting buffer.
  • the spotting buffer composition may be varied to accommodate particular experimental or manufacturing parameters.
  • the spotting buffer may comprise, for example, an appropriate buffer including but not limited to 20mM- 200mM Tris, pH 7.0-9.0, a non-ionic surfactant such as Zonyl® FSN (E.I. DuPont DeNemours & Co., Cat. No. CH 7152S) in a concentration range of 0.1%-1.0% (weight/volume) bovine serum albumin (BSA) at 0.5%-4.0% and 0.1% sodium azide.
  • BSA bovine serum albumin
  • the spotting buffer comprises 30-100 mM Tris, pH 7.0-8.5, 0.1%-0.5% Zonyl® FSN, 1.0%-3.0% BSA and 0.1% sodium azide.
  • the spotting buffer comprises 50mM Tris, pH 8.0, 0.1% Zonyl® FSN, 2.0% BSA and 0.1% sodium azide.
  • Fluorinated surfactants e.g. 3M Cat. No.'s FC 171 and FC 170C
  • other appropriate surfactants known to the skilled artisan may be substituted for Zonyl® FSN.
  • Tris-buffered solution pH 7.5 including BSA, stabilizer and 0.1% sodium azide as a preservative.
  • Calibrator solutions and the selected cross-linked reagent varied depending upon the analyte tested.
  • the hTSH assay is performed on the Stratus® or Stratus® II instrument by aspirating and delivering 60 ⁇ l of a selected calibrator (or sample) onto a tab that contains an optimum quantity of the cross-linked antibody complex.
  • the Stratus® instrument substrate probe then aspirates
  • the substrate wash solution is added in 20 ⁇ l and 50 ⁇ l aliquots to the tab. Depending on the assay chosen, the volumes of different reagents applied may differ. As soon as the second substrate wash aliquot is delivered, the initial fluorescence rates are read and recorded in the instrument memory.
  • the amount of fluorescence generated by action of, for example, alkaline phosphatase enzyme label on a methylumbelliferyl phosphate substrate is detected by the Stratus® instrument and converted to a "rate" expressed in voltage per unit time, which is presented in the Tables and Figures as mV/min ("Stratus Rates").
  • the Stratus rate is a measure of the intensity of the fluorescence, which is, in turn, a measure of the amount of analyte or other reactive substance bound to the reactive portion of the tab.
  • R ⁇ ( A - D) / [ 1 + B (X/C)] + D ⁇
  • R the fluorescence rate
  • X the corresponding concentration.
  • the equation is a generalized sigmoidal curve that has been reported to give an excellent fit in various immunoassay systems. Based on the resulting A, B, C and D parameters stored in the memory, the instrument provides the concentration readout for the samples assayed.
  • EXAMPLE 3 Cross-linked Anti-CEA Antibodies
  • Purified anti-carcinoembryonic antigen (CEA) monoclonal antibody (clone # CEL007, Catalog # 200088; Hybritech, Inc.) was buffer- exchanged into 0.1 M sodium phosphate (pH 7.0) and the volume reduced so that the target antibody concentration was about 6.5 +/-0.5 mg/mL.
  • Antibody concentrations were estimated spectrophotometrically using an extinction coefficient at 280 nm of 1.48 mg(cm-im L -i ) .
  • Bis(sulfosuccinimidyl) suberate (BS3) (Pierce Chemical Co., Cat.
  • the protein component was separated from small molecular species by passage through a Sephadex G-25 column (Pharmacia, Cat # 17- 0033-02) that had been equilibrated in phosphate buffered saline (PBS) containing 0.1% NaN 3 (i.e. 0.1 g NaN 3 /100 ml PBS).
  • PBS phosphate buffered saline
  • the protein component was purified by using a P-6 DG column (Bio-Rad, Cat #
  • the concentration of the cross-linked antibody was determined by a BCA protein assay (Pierce, Cat. No. 23225G). Antibody solutions were adjusted to give final protein concentrations of 100 to 600 ⁇ g/mL.
  • CEA spotting buffer 50mg sodium phosphate, 0.3 M sodium chloride, 0.1% Tween 20, 1% BSA, 2% potassium gluconate (pH 6.90)). About 76 ⁇ L of the various concentrations of protein solutions were spotted onto blank tabs and dried in an oven at about 80 °C .
  • a control tab was prepared using the traditional double immunocomplex assay format.
  • the double immunocomplex was formed by mixing about 190 ⁇ g/mL primary monoclonal antibody with optimized amount of secondary antibody, i.e. goat anti-mouse polyclonal (GAM) antibody, and spotted onto a glass filter tab.
  • Calibrated standards, Cal A to Cal F contained 0.0, 0.25, 0.75, 3.0, 12.0 and 50.0 ng/ml CEA respectively.
  • Table 1 is a comparison of results obtained with cross-linked tabs and with the double immunocomplex control tabs.
  • the protein concentration was determined by BCA protein assay.
  • the protein was then placed in TFSSB (50mM Tris, 2% BSA, 0.1% gelatin, 0.15 M NaCl, 0.1% FSN (zonyl fluoro-surfactant) (pH 8.0)).
  • TFSSB 50mM Tris, 2% BSA, 0.1% gelatin, 0.15 M NaCl, 0.1% FSN (zonyl fluoro-surfactant) (pH 8.0)
  • Several concentrations of the cross-linked protein complex were prepared and spotted on glass filter tabs for use in the Stratus® instrument.
  • Calibrators A, B, C, D, E, and F used in the assay contained 0.0, 0.5, 1.5, 15.0 and 50.0 ⁇ lU/ml of hTSH.
  • Fig. 1 depicts the Stratus® A, B and F rates as functions of increasing concentrations of cross-linked D70 antibodies.
  • Table 2 is a comparison of TSH assays using tab
  • Cal B represents the first measurable quantity of the analyte.
  • Cal A zero analyte
  • Cal F represents the highest amount of the analyte to be detected.
  • the ratio Cal B/Cal A is a measure of low end sensitivity and the ratio Cal F/Cal A represents overall sensitivity.
  • the higher calibration curve ratios for the cross-linked tabs indicate an increased sensitivity for the assay using cross- linked tabs compared to the assay using the double immunocomplex tabs.
  • Table 3 is a direct comparison of Stratus® rates and the Rodbard parameters for the TSH assay using both cross-linked D70 tabs and double immunocomplex tabs.
  • Cross-linked D70 tabs made from 150 ⁇ g/mL cross -linked protein complex
  • reaction was quenched by the addition of 1 M ethanolamine (pH 8) equal to a 1/9 volume of the reaction mixture and the incubation was continued at room temperature for 0.5 hours. Then the reaction mixture was purified by passing through an Ultrogel AcA-44 column (IBF Biotechnics, Cat # 230161). After protein concentration determination by
  • the cross-linked anti-hTSH was placed into TFSN (50mM Tris, 2% BSA, 0.1% FSN, 0.1% NaN 3 , pH 8.0) containing 0.02% poly-L-lysine (Sigma, Cat # P-7890).
  • TFSN 50mM Tris, 2% BSA, 0.1% FSN, 0.1% NaN 3 , pH 8.0
  • poly-L-lysine Sigma, Cat # P-7890
  • Figures 2 and 3 show the results obtained with a Stratus® TSH assay using glutaraldehyde-crosslinked CA2, compared to the traditional double immunocomplex tabs (CA2:GAM) on Stratus® and Stratus® II, respectively.
  • Calibrators A, B, C, D, E, and F used in the assay contained 0.0, 0.25, 0.75, 3.00, 12.00 and 50.00 ⁇ lU/ml of hTSH.
  • Raw data for the figures including the Rodbard instrument parameters are given in Table 4. Table 4
  • Table 4 shows comparative performance of hTSH tabs prepared using cross-linked CA2 and tabs prepared from a double- immunocomplex on Stratus® and Stratus® II. Signal readings generated for the different calibrations using cross-linked tabs in Stratus® and Stratus® II is shown in columns 3 and 5 respectively. Corresponding data for the double-immunocomplex are shown in columns 2 and 4, respectively.
  • Cross-linked Anti-TAT Ffab > 2
  • a capture reagent consisting of an F(ab') 2 fragment of antibody against thrombin:antithrombin complex (Anti-TAT F(ab') 2 ) was buffer-exchanged into 0.1 M sodium phosphate (pH 7.6) and the volume was reduced so that the final F(ab') 2 concentration was about 5.00 +/- 0.25 mg/mL.
  • the cross-linked F(ab') 2 was purified by passing through a Sephadex G-25 column.
  • the concentration of cross-linked F(ab') 2 was determined either by BCA protein assay or spectrophotometrically using an extinction coefficient of 1.48 mg/(mL)(cm).
  • An ELISA was performed using the cross-linked F(ab') 2 .
  • the plates were first coated with the cross-linked F(ab') 2 overnight at 2-8°C.
  • TAT calibrators (0-50 nM) were added and the plates were incubated at ambient temperature for 0.5 hour.
  • Anti-TAT antibody-HRP horseradish peroxidase conjugate was added and incubated at ambient temperature for 15 minutes followed by the addition of the substrate. The incubation then was continued for additional 15 minutes at ambient temperature.
  • the plates were washed twice with a wash buffer before each new reagent was added. The plates were monitored at 405 nm using a plate reader in order to determine the colorimetric readout.
  • Table 5 is a comparison of ELISA performance between cross- linked F(ab') 2 plates and non-cross-linked F(ab') 2 plates.
  • This table shows the signal generated in the presence of calibrators F (50 nM) and A (0.0 nM) as measured by absorption at 405 nm in the ELISA assays using untreated F(ab') 2 and cross-linked F(ab') 2 -
  • overall sensitivity ratio Cal F/Cal A
  • This increase in sensitivity for the cross-linked antibody plates was not only due to decrease in non-specific binding (lower signal for Cal A) but also due to higher response for the Cal F resulting from more efficient immobilization of the cross-linked antibody on the plates.

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Abstract

L'invention concerne des procédés et des compositions servant à réaliser des titrages par liaison spécifique, dans lesquels des réactifs de liaison spécifique sont immobilisés sur une phase solide. L'immobilisation est facilitée par couplage de réactifs bifonctionnels à des protéines de titrage par liaison spécifique, ce qui réticule chimiquement les réactifs de titrage par liaison spécifique. Ces réactifs réticulés permettent de réaliser des titrages sans avoir recours à des réactifs secondaires. Cet avantage permet d'augmenter la sensibilité du procédé en limitant l'interférence d'arrière-plan non spécifique avec la phase solide ou avec les spécimens cibles.
EP94928017A 1993-09-22 1994-09-07 Immobilisation de proteines chimiquement reticulees sur des supports solides Withdrawn EP0670040A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US12537893A 1993-09-22 1993-09-22
US125378 1993-09-22
PCT/US1994/010032 WO1995008771A1 (fr) 1993-09-22 1994-09-07 Immobilisation de proteines chimiquement reticulees sur des supports solides

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EP0670040A1 true EP0670040A1 (fr) 1995-09-06

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EP (1) EP0670040A1 (fr)
JP (1) JPH08509064A (fr)
AU (1) AU7721094A (fr)
CA (1) CA2150029A1 (fr)
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EP1387692A4 (fr) * 2000-11-11 2004-07-21 Arbor Vita Corp Interactions moleculaires dans des cellules hematopoietiques
EP2339342B1 (fr) 2004-09-14 2014-11-12 LSI Medience Corporation Biomatériau, procédé de fabrication et applications de celui-ci
JP5458344B2 (ja) * 2008-01-30 2014-04-02 旭化成株式会社 抗体固定化担体

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US4517288A (en) * 1981-01-23 1985-05-14 American Hospital Supply Corp. Solid phase system for ligand assay
US4486408A (en) * 1981-04-07 1984-12-04 Kiel Johnathan L Insoluble crosslinked cytotoxic oxidase-peroxidase system
US4714676A (en) * 1982-09-16 1987-12-22 Owens-Illinois Glass Container Inc. Protein modification to provide enzyme activity
AU626809B2 (en) * 1987-10-30 1992-08-13 Abbott Laboratories Heterobifunctional coupling agents

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JPH08509064A (ja) 1996-09-24
WO1995008771A1 (fr) 1995-03-30
CA2150029A1 (fr) 1995-03-30
AU7721094A (en) 1995-04-10

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