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WO1992002818A1 - Dosage immunologique a addition sequentielle sur une matrice - Google Patents

Dosage immunologique a addition sequentielle sur une matrice Download PDF

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Publication number
WO1992002818A1
WO1992002818A1 PCT/US1991/005694 US9105694W WO9202818A1 WO 1992002818 A1 WO1992002818 A1 WO 1992002818A1 US 9105694 W US9105694 W US 9105694W WO 9202818 A1 WO9202818 A1 WO 9202818A1
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Prior art keywords
matrix
analyte
binding protein
analog
protein
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PCT/US1991/005694
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English (en)
Inventor
Linda J. Janis
Fred E. Regnier
Noubar B. Afeyan
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Purdue Research Foundation
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Publication of WO1992002818A1 publication Critical patent/WO1992002818A1/fr

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    • 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
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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
    • 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
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G01N33/561—Immunoelectrophoresis

Definitions

  • the present invention is generally related to a method and apparatus for the assay of an analyte. More particularly, this invention relates to a rapid method for the quantitative assay of plural samples of analytes useful as a monitor of product quality during protein production.
  • product quality is a function of both purity and structure. Once expressed, the protein must be separated from all other cellular components, many of them present in excess of the protein of interest and/or having similar purification profiles. For example, if the product is a therapeutic substance for human use, federal regulations require all contaminating components be removed to a level of less than 1 ng/dose of compound. This generally requires that the product be of at least 99.9% purity.
  • the native conformer must be separated from all variants of the protein which may arise as a result of the purification process or errors in biosynthesis. It is known that organisms which have been induced to express a protein can misincorporate amino acids at certain positions in the protein. In fact, up to 5% of the proteins produced can have such expression errors. Other biosynthetic errors also may be introduced during post-translational modification of the protein, including improper glycosylation, proteolysis, or incorrect disulfide bond formation, all of which can affect the protein's three dimensional structure.
  • Conformational changes to the protein are important because, in general, it is the native conformer of the protein that is biologically active. In addition to diminishing the biological activity of proteins, structural alterations in the protein may increase the protein's immunogenicity. This is particularly undesirable in therapeutic proteins that must be administered repeatedly.
  • High performance liquid chromatography HPLC
  • HPCE high performance capillary electrophoresis
  • RAIA rapid analytical immunoassay
  • Chromatography and electrophoresis analyze samples by separation, based on size or charge. During the early stages of purification the product may be contaminated with a large number of other proteins and constitute less than 10% of the total protein mass, making it difficult to identify the protein of interest by conventional separation techniques. Similarly, at the very late stages of protein purification, protein purity is very high, generally exceeding 95%.
  • a chromatogram or electropherogram is essentially a single peak, making it difficult to detect small concentrations of contaminants in this range. Separation methods here are also of limited utility.
  • chromatographic and electrophoretic analyses are limited by the time required to perform their protocols.
  • the speed of HPLC analysis generally is limited by the porous chromatographic packings used in conventional HPLC chromatography. While mobile phase mas& transfer between the matrix particles is convectional in HPLC chromatography, mobile phase mass transfer within the porous particles is primarily diffusional, substantially slowing the progress of the protein through the matrix.
  • the speed of electrophoresis separation is a function of the voltage applied across the system. However, heat production increases with the square of the voltage. Thus, the maximum voltage most electrophoresis systems will tolerate is limited by the speed with which they can dissipate the heat generated by the applied voltage.
  • rapid analytical immunoassay appears to be the only technique of sufficient discriminating power to isolate a protein of interest from both crude samples and substantially pure products.
  • an immunoassay involves a specific binding interaction with the protein of interest, to a first approximation, the assay provides a method of determining if the protein is folded correctly.
  • conventional analytical immunoassays performed as equilibria- reactions, also are limited by the incubation times required in most assays. Immunoassays are based on the well-known reactions between an antibody and the specific antigen or hapten (analyte) for that antibody, although the general principal can be applied to any specific binding interaction between a binding protein and its ligand.
  • an analog of the sample antigen or analyte competes with the sample antigen for complex formation with an antibody specific for that antigen.
  • the competing analog is often a purified form of the sample antigen that is tagged, e.g., with a fluorescent or radioactive tracer material.
  • the analog also can be tagged with an enzyme having a readily assayable activity, such as reacting with a substrate to give a colored product.
  • the fraction of tagged analog bound tothe antibody (or remaining free in solution) is then measured, and is related to the amount of sample antigen (analyte) present in the reaction. It has generally proven easier to quantitate the bound species, as this species is often the easier of the two to collect.
  • SAIA sequential addition immunoassay
  • SAIAs have been shown to be several times more sensitive than simultaneous incubation assays for a given concentration of sample antigen, as they allow all target antigen an opportunity to bind antibody before the addition of a competitor.
  • Bound complexes can be collected by precipitation, usually with a second antibody (so called “sandwich” or “double antibody” assays.)
  • a second antibody so called “sandwich” or “double antibody” assays.
  • one of the components can be immobilized on an easily separable solid support, such as a test tube wall or Sepharose beads.
  • Immobilized immunoassays comprising Sepharose beads are particularly attractive for analytical assays because the beads, can be packed into a chromatography column, allowing the sample to be captured, and impurities to be washed away.
  • the antibody (or immobilized component) is generally covalently attached to the solid support.
  • the antibody may interact reversibly with the solid support if the support's surface includes a component, such as protein A, capable of specific, reversible interaction with the antibody. If the immobilized component is reversibly bound to the solid support, the bound complexes can be eluted and the analog-antibody complexes quantitated. Quantitation requires that the analog be tagged in some way, to distinguish complexed analog from complexed sample antigen. See, for example, Mattiason et al., Proc. Int. SYTOP. on Enzyme-Labelled Immunoassay of Hormones and Dru ⁇ s, Pal, S., Ed., Walter de Gruyter, Berlin (1978), p. 91.
  • the sample antigen can be analyzed by simply eluting it from the column and quantitating it with a UV detector, potentially eliminating the need for a tagged competitor.
  • many compounds used to elute sample antigens, so-called eluting agents coelute with the antigen and can cause a significant perturbation of the detection base line, making it difficult to differentiate the elution profile of the sample antigen from that of the eluting agent. In practice, this effect can diminish the sensitivity of the assay by more than 100 fold, depending on the detection wavelength and eluting agent used.
  • a second disadvantage with this system, and of particular importance for a rapid monitoring system, is that it requires covalent immobilization of the antibody or binding protein to prevent its elution with the antigen. This means that the column must either be dedicated to the analysis of a single antigen for its lifetime, or that the column be repacked each time a different sample antigen is to be sampled.
  • Binding protein includes any sample compound to be analyzed that is capable of being bound specifically and reversibly to another compound, herein referred to as the "binding protein".
  • useful analytes and bindin ⁇ proteins include antigens (or haptens) and antibodies, hormones and receptors, arid any other compound combinations capable of specific binding interactions that are essentially reversible, (preferably having a dissociation constant less than about 10" 8 M) .
  • Analog includes any compound capable of binding to the binding protein in a way analagous to analyte binding.
  • the analog includes compounds that are the same as the analyte, as well as analogs of the analyte, such as those that have different binding affinities or that are tagged in some way (as with a fluorophore or radioisotope) .
  • Zero sample dose describes an assay run in the absence of sample analyte.
  • the amount of free analog exiting the matrix at zero sample dose is defined as F Q .
  • Free analog is the amount of free analog exiting the matrix unbound in a given assay.
  • the amount of free analog exiting the matrix is defined as F.
  • Dose response curve is the standard curve plotting the amount of free analog exiting the matrix for a range of analyte standard concentrations, for a given initial concentration of analog and binding protein, and for a given flow rate.
  • the "fraction of free analog” (F/F Q ), as used herein, is the ratio of free analog (F) to the amount of analog exiting the matrix at zero sample dose .
  • Matrix as used herein, means a structure defining a surface area accessable to analyte and analog flowed through or by the surface and having binding protein immobilized thereon. It is an object of this invention to provide a method and apparatus for the rapid, quantitative assay of an analyte that can be used as part of a protein production monitoring system for all stages of protein purification, and which can be performed under non-equilibrium conditions. Another object of the invention is to provide a method and apparatus for the sequential assay of plural, different samples of analytes. Still another object is to provide a method for the rapid assay of plural samples of an analyte to overcome the time limitations imposed by conventional immunoassay and separation techniques.
  • This invention features a method for the assay of an analyte utilizing a matrix to which is attached binding protein having binding sites for an analyte, and for an analog.
  • the method can be used to assay the quantity of analyte in a given sample by simply measuring the amount of free analog exiting the matrix.
  • the method is rapid and quantitative and can be used to assay different analytes, or to assay plural samples of one analyte.
  • the method comprises the steps of providing a matrix having a known quantity of a binding protein capable of binding the analyte specifically attached to it
  • the binding reaction should be essentially irreversible, preferably having a K ⁇ 3 of less than about 10- 8 M.
  • the quantity of binding protein used for a given assay is such that the binding protein occupies only a small portion of the available attachment sites on the matrix, i.e., less than 20%, and more typically less than 1.0%.
  • a preselected volume containing the analyte is then added to the matrix. The amount of analyte in the volume should be insufficient to saturate the binding sites of the binding protein.
  • the binding protein specifically binds the sample analyte in the volume, thereby "capturing" the analyte while all other solutes present in the sample volume pass through the matrix.
  • a known amount of analog is then added to the matrix. The amount added is at least equal to the total number of binding sites on the matrix.
  • the amount of free analog (F) exiting the matrix is then quantitated and is related to the amount of analyte bound by comparison to a standard curve derived for a given flow rate, the ratio F/FQ being directly related to the amount of analyte bound in the assay.
  • the assay is preferably performed under non-equilibrium conditions, allowing rapid measurement without a concomitant loss of sensitivity, and does not require quantitation of bound components, substantially eliminating the problems associated with desorption kinetics and desorption buffer interferences. Moreover, because the analog is added after the sample antigen, unbound analog will exit the matrix free of any non-analyte solutes present in the sample volume. Hence, the analog need not be tagged to be identified.
  • the assay may be run as a simultaneous assay, where both the analyte and analog are added to the matrix at the same time.
  • the analog must be tagged to identify it, as other solutes, including some target analyte, may coelute with free analog.
  • the sensitivity of a simultaneous assay can be further enhanced by utilizing an analog havi ⁇ .n a lower binding affinity for the binding protein than the target analyte. In this way, more target analyte will have an opportunity to be captured as it passes through the matrix and will be less likely to pass through unbound.
  • This type of analog also may be used in sequential assays.
  • the method of this invention may be used for the rapid, quantitative assay of plural samples of an analyte, or for the rapid, quantitative assay of different analytes.
  • the system is easily regenerated after each assay by eluting both the binding protein and bound analyte/analog from the matrix, and reloading the matrix with fresh binding protein.
  • a matrix surface that can be regenerated easily provides a flexible protein monitoring system readily adapted to analyze different samples.
  • the system need not be recalibrated between assays if all the binding protein added to the matrix in each assay will be bound by the matrix.
  • the binding protein should be present at a fraction of the matrix's binding capacity for the binding protein in each assay.
  • Another requirement for reliable analysis of repeated assays is that the volume of the system remain as small as possible. Repeated use of the matrix may decrease its attachment surface capacity over time as the matrix surface breaks down. If a large volume system is used, reactions may begin to occur in isolated sub-areas or "reaction zones". The apparent binding protein concentration available within a reaction zone then may differ from the total binding protein concentration added, and the data generated may become unreliable.
  • the requirements for a high binding protein attachment surface area and a small volume can be achieved with a chromatography system using a porous matrix, such as provided in conventional HPLC matrix material, and transporting solutes through the matrix along a pressure gradient.
  • a 500 A pore size, 30 ⁇ m particle size matrix material in a 0.4 ml volume column, for example, provides sufficient surface area so that each assay using 10 ⁇ g of binding protein uses only 1/25 - 1/30 of the total matrix binding capacity.
  • chromatographic matrices capable of perfusive chromatography.
  • These matrices comprise particles which may be of the same overall size as are sometimes employed in conventional matrices, but having increased intraparticle porosity.
  • particles capable of perfusive chromatography have a network of 500-1500 A pores interconnecting the larger throughpores. The resulting network limits the diffusional path lengths within the particles so that mass transfer within the particle pores is essentially governed by convection.
  • the ability to use high flow rates without a substantial loss of resolution using a perfusive chromatography system allows the assays of this invention to be run at high speed without requiring increased quantities of binding protein.
  • the reduced requirement of binding protein per assay can increase the useful life of the matrix.
  • the increased porosity of the particles capable of perfusive chromatography substantially increases the surface area available for attachment of binding protein, typically to levels within the range of 30 to 50 m 2 /ml.
  • One ml of perfusive matrix is capable of binding approximately 10-20 mg of binding protein.
  • the fraction of available attachment sites occupied in an assay using 10 ⁇ g of binding protein occupies less than 1/1000 of the total binding protein attachment capacity of the matrix.
  • binding protein would be present at a surface density of less than 3 ⁇ g/m 2 . Quantities of binding protein less than 10 ⁇ g will occupy an even smaller portion of the available attachment sites.
  • the exceptionally high surface area provided by perfusive mr 1 rices allows one to reduce the column volume required for an assay significantly while still maint ⁇ .ining a large excess of binding protein attachment surface area, essentially eliminating concerns about the occurrence of reaction zones over time.
  • the system can be regenerated after each assay, providing a flexible, reusable assay system.
  • sequential samples of the same analyte or of different analytes may be measured by this method, without requiring recalibration of the system between assays.
  • the invention involves a method for the rapid, quantitative sequential assay of plural samples of analytes.
  • An assay comprises the steps of reversibly attaching a binding protein having binding sites for an analyte to a matrix such that the amount of binding protein attached to the matrix occupies a small portion of the available attachment sites on the matrix, performing a quantitative assay using the analyte binding sites on the matrix to bind the analyte from a sample volume, and then removing the binding protein from the matrix.
  • the system is now regenerated using a second binding protein, which may have binding sites for a second, different analyte.
  • Another method of achieving the high surface area/low volume requirements of this invention is to use high performance capillary electrophoresis.
  • the binding protein is coated onto the inner surface of a capillary tube (e.g., diameter less than 100 urn), at a surface density such that binding protein will occupy only a fraction of the available surface for any given assay. Solutes then are transported through the capillary tube matrix along a voltage gradient created when a voltage is applied across the system.
  • the surface area to volume ratio of these capillary tubes is significantly greater than that of other electrophoresis systems. This high ratio provides the system with excellent heat dissipation properties, allowing one to apply larger voltages across the system, significantly reducing assay time.
  • another aspect of the invention involves a method and apparatus for the rapid assay of analytes where the analyte is transported along a voltage gradient.
  • the binding protein/analyte combination can be any protein combination capable of specific reversible binding interactions.
  • the binding protein and analyte comprising any enzyme/substrate combination, including receptor and ligand, or antibody and antigen.
  • the binding protein can be reversibly bound to the matrix surface.
  • the matrix surface may comprise an antibody specific to the Fc region of the binding protein antibody (such as immunoglobulins generated cross-species) .
  • protein A or protein G two proteins known to bind to the Fc region of immunoglobulins may be used.
  • preferred matrices have protein A or protein G attached to their surface. Non-specific adsorption of binding protein directly onto the surface of a chromatography matrix also may be used.
  • Analogs exiting the matrix may be detected in a number of different ways.
  • free analog exits the matrix without contaminating coeluents and therefore does not need to be tagged to be identified.
  • the analog In the case where the analog is a protein, one can quantitate unbound analog exiting the matrix by measuring UV absorbance at 225 nm with a spectrophotometer.
  • the analog may be tagged with a detectable moiety. If the reaction is run as a simultaneous assay, the analog must be tagged.
  • a currently preferred tag is a fluorophore, although other commonly used tags, such as.enzyme tags, may be useful.
  • a fluorophore can be detected with a spectrofluorimeter, or a spectrophotometer, measuring absorbance at 495 nm.
  • SAIAs run with fluorescently tagged analogs also provide an internal "self check" mechanism to verify that other proteins are not coeluting with the analog. Only the analog will be detected at 495 nm, but all proteins present in the eluent will absorb at 225 nm.
  • the apparatus of the invention may provide a means of calculating an displaying the amount of analyte present in the analyzed sample.
  • FIGS. 1A and IB are schematic diagrams illustrating methods of the invention
  • FIG. 2 is a schematic representation of an apparatus embodying the invention.
  • Figures 3, 4, 5 and 6 are graphs illustrating various principles and characteristics of the present invention.
  • this invention describes a method and apparatus for the rapid assay of an analyte.
  • the process is particularly useful as a monitor of protein quality in a protein production process.
  • a matrix 10 is provided having on its surface attachment sites 12 capable of specific and reversible interaction with a binding protein 14 having binding sites 16 for the analyte to be measured.
  • the quantity of binding protein 14 added to the matrix is a small fraction of the attachment sites 12 available for attaching the binding protein to the matrix. This ensures that all the binding protein added interacts with attachment sites on the matrix to form a complex 18.
  • a volume containing the analyte 20 to be sampled, and optionally, other non-analyte solute components 22, is then flowed through the matrix system.
  • the amount of the analyte added must be insufficient to saturate the binding protein binding sites 16.
  • the analyte 20 interacts specifically with these binding sites 16 to form a complex 24, while non-specific components 22 pass through the matrix unbound.
  • an analog 26, identical or analogous to the sample analyte is added to the matrix system in an amount at least equivalent to the number of the binding sites 16 on the matrix.
  • the sequential assay of this invention is most useful when performed under nonequilibrium conditions. Provided flow rates are uniform in calibration runs and analysis runs, there is no requirement that the binding reactions reach equilibrium. As such, the amount of analyte-binding protein complex formed for any given assay is dependent on the concentration of the reactants and the length of time that the reaction is allowed to progress. Accordingly, the system provides the experimenter with a broad experimental range, as the experimenter can vary both the concentration of the reactants and the duration of the experiment, as desired.
  • a calibration curve can be determined by preparing a standard dose response curve using samples having a range of analyte concentration, using a given binding protein matrix, a given analog concentration, and a given flow rate.
  • the standard curve can be determined experimentally or, where the rate constants for the reaction are known, by calculation.
  • the amount of analog exiting the matrix at zero sample dose (F Q ) can be measured experimentally, or extrapolated from the standard curve.
  • reaction time flow rate
  • choice of flow rate is not critical, as the amount of free analog exiting the matrix will always be a function of the amount of analyte bound. While a slower flow rate (longer reaction time) allows a greater fraction of analog to bind, it is not required for quantitation.
  • analog 26 must be tagged to differentiate it from sample analyte 20 and other, non-specific compounds 22.
  • the analog has a lower binding affinity for the binding protein than the analyte, thereby allowing more analyte to bind, and providing a more sensitive assay.
  • Sequential assays made on a product stream from a protein production system or on other types of protein solutions can be carried out using an apparatus such as that depicted schematically in Figure 2.
  • the apparatus preferably comprises a multi-port sampling valve 28 such as is found in automated protein production systems known to those skilled in the art, and which allocates the various components of the assay to matrix 40, whose surface is capable of reversibly binding the binding protein.
  • reservoirs containing buffer 30, binding protein 32, analog 36, and a recycling solvent 38, and means for obtaining a sample 34, e.g., an accumulator, controlled by valve 28, provide fluids to matrix 40, impelled by, e.g., pump 31.
  • the recycling solvent 38 is capable of desorbing all reversibly bound complexes from the matrix surface.
  • Valve position of the multi-port sampling valve 28 is preferably under computer control.
  • buffer delivery to the matrix may be driven by a metering pump 29. Effluent exiting the matrix is either discarded through line 42, or quantitated in a detector, 44, e.g., a conventional detector which measures U.V. absorbence through a film of fluid, and produces a curve which, when integrated, gives quantitative information.
  • the apparatus may further comprise a means 46 for calculating analyte concentration and for displacing data indicative of the amount of analyte in the sample.
  • the column 40 is maintained either at equilibrium with buffer from reservoir 30, or preferably in a rest state in which it is loaded with binding protein from reservoir 32.
  • the system is activated to cycle through a series of steps involving flowing at a predetermined flow rate a metered quantity of sample 34, followed by a metered quantity of analog 36.
  • analog 36 is distinguished from the sample by the detector 44 (e.g., if the analog is t ⁇ ged)
  • the sample and analog may be passed through matrix 40 together. Effluent from the column produced by the breakthrough of sample 36 is measured for protein concentration by detector 44 .
  • the amount of analog detected is then correlated to analyte concentration by, for example, electronic comparator means in module 46.
  • HTr Human transferrin
  • Serum samples containing HTr were obtained from St. Elizabeth's Hospital, Lafayette, IL. Pure HTr was purchased from Sigma Corp., St. Louis, MO.
  • Rabbit anti-HTr was used as the binding protein, (Boehringer Mannheim Biochemicals, Indianapolis, IN), obtained as an IgG fraction of antiserum.
  • a conventional protein A column was used as the reversible matrix binding surface (Chromatochem, Inc., Missoula, MT) . Protein G columns also were used to verify several principles of the invention.
  • HTr was labeled with fluorescein isothiocyanate, Isomer I (FITC, 10%, on Cellite) using the method of Rinderknect (Nature 152:167 (1962)) for the simultaneous incubation studies. Briefly, HTr was dissolved in 0.05 M sodium carbonate (pH 8.5). To this solution was added approximately 15 mg of Cellite, containing 10% FITC (Sigma Co., St. Louis, MO). The mixture was shaken for 4 minutes, and then centrifuged for 3 minutes. This treatment was sufficient to transfer label to the protein, and the FITC-labeled protein was purified by column chromatography (Sephadex G25, in 0.01 M phosphate, 0.15 M NaCl, pH 7.0 buffer). The FITC to protein ratio (F/P) was determined from the concentration of protein and FITC obtained from absorbance at 280nm and absorbance at 495nm (The, et. al.. Immunology l ⁇ :865 (1970)).
  • the binding protein (AHTr) capacity of the protein A affinity chromatography matrix was evaluated by frontal analysis (Jacobson et al., J. Chromatoor. 3_1£:53 (1984)). Duplicate 3.5 ml samples of a 0.25 mg/ml solution of AHTr in a buffer containing 0.01 M sodium phosphate, 0.15 M NaCl (pH 7.0) was loaded onto the affinity column at 0.5 ml/min until a breakthrough curve appeared. AHTr was desorbed with 0.1 M glycine, 2% acetic acid (pH 2.9).
  • the binding protein capacity of this column was determined to be 0.51 mg AHTr. Thus, a given assay using 10-20 ⁇ g will only use about l/25th of the column's capacity.
  • Nonspecific binding of HTr to protein A or protein G columns was measured by successive injections of HTr (1 ⁇ l, 2 mg/ml). Five successive injections yielded constant peaks for each injection, indicating that nonspecific binding of HTr to the affinity column was very low. Nonspecific binding would have resulted in peak areas slowly increasing with each injection until a maximum was reached.
  • Loading buffer for all analyses was 0.01 M sodium phosphate, 0.15 M NaCl (pH 7.0).
  • AHTr (2 ⁇ l) was injected into the protein A column at the same flow rate (2 ml/min.) for all analyses. This amount of AHTr will theoretically bind up to 4 ⁇ g of HTr, calculated from the manufacturer's reported titer (2 ⁇ g HTr/ ⁇ l AHTr). The flow rate was then adjusted to 1 ml/min and either 1 ⁇ l injections of HTr standards or aliquots of 1 ⁇ l of human serum were injected into the affinity column.
  • tagged analog pure HTr, 1 ⁇ l, at 4 ⁇ g/ ⁇ l
  • the peak area of the free analog exiting the matrix was measured.
  • antigen-antibody complexes were desorbed from the column at 2 ml/min., using 2 ml of desorbing agent. The column was then re-equilibrated.
  • the method was essentially the same as for SAIAs except that antigen standards (or human serum) and FITC-labeled HTr (1 ⁇ l each) were mixed in the needle of the autoinjector prior to injection into the protein A column.
  • the chromatogram of a typical SAIA is shown in Figure, 4. Eluents were measured by UV absorbance at 225 nm and the free fraction of analog quantitated by peak area.
  • the peak area labelled A comprises nonspecific eluents from the antiserum IgG fraction.
  • the peak labelled B indicates eluted nonspecific components from the serum sample.
  • Peak C contains unbound analog eluting from the matrix.
  • peak D contains all bound complexes desorbed from the matrix surface.
  • a number of desorbing agents were tested for their ability to remove antibody-antigen complexes from the protein A columns.
  • Protein A and immunoglobulins have two types of interactions: hydrophobic and ionic.
  • the most effective desorbing agent found so far has proven to be a 50% ethylene glycol/10% acetic acid solution, as the ethylene glycol helps to dissociate the hydrophobic interaction, and the acetic acid affects the ionic interaction. Twenty percent acetic acid has also been used, as have 0.1 M glycine and 2% acetic acid solutions.
  • the absorbance spectrum of a typical, simultaneous incubation immunoassay using FITC-HTr is shown in Figure 5.
  • the free fraction of analog was quantified by peak area measured at 495 nm.
  • the 280 nm peak measured the absorbance of all unbound protein components, including analog, eluting from the matrix.
  • Figure 6 compares theoretical and experimentally derived dose response curves for four different flow rates: (A.) 2.0 ml/min; (B.) 1.0 ml/min; (C.) 0.5 ml/min; and (D.) 0.1 ml/min. As illustrated, in all cases the curves corresponded remarkably well.
  • the theoretical curves were derived using numerical solutions for the rate equations as described in Rodbard et al., (1971, J. Clin. Endocrinol. , 3.2.:343-355) .
  • Binding protein volume was assumed to be the void volume of the column, and sample and analyte volumes were calculated from the peakwidth at half height on chromatograms. Time was described as the ratio of the column void volume to flow rate.
  • the experimental curves were determined by conducting multiple assays using the procedure disclosed herein.
  • flow rates of 1 ml/min were found to be optimal. Optimization of the assay using conventional HPLC matrices can decrease the time required to run each cycle to approximately five minutes. Use of a perfusive matrix can increase the throughput speed of the assay substantially. In addition, use of a perfusive matrix, with its exceptionally high surface area, can also increase the dynamic range of the assay.

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  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Procédé de dosage quantitatif rapide d'un analyte. Ledit procédé utilise le flux à travers une matrice, par ex. une colonne de chromatographie, ayant des sites de fixation pour une protéine de liaison qui est capable de lier spécifiquement l'analyte et un analogue. Ledit dosage est effectué de préférence sous forme de dosage immunologique séquentiel dans des conditions de non-équilibre. L'analogue ne nécessite pas d'être marqué pour être identifié et la quantité d'analogue libre quittant la matrice est directement en rapport avec la quantité d'analyte lié à la matrice. La liaison entre les sites de liaison de la matrice et les protéines de liaison peut être réversible, ce qui permet au système d'être régénéré de novo pour chaque dosage.
PCT/US1991/005694 1990-08-10 1991-08-09 Dosage immunologique a addition sequentielle sur une matrice WO1992002818A1 (fr)

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US565,628 1990-08-10

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EP0707211A1 (fr) * 1994-10-15 1996-04-17 BEHRINGWERKE Aktiengesellschaft Phase solide réutilisable pour les réactions des liaisons spécifiques
WO1999013330A3 (fr) * 1997-09-08 1999-06-17 Roche Diagnostics Gmbh Epuration de substances provenant d'un echantillon biologique
WO1999036776A1 (fr) * 1997-12-30 1999-07-22 Pharmacia & Upjohn Diagnostics Ab Procede analytique comprenant une addition dans deux ou plusieurs positions et dispositif et trousse d'analyse associes
WO2001022078A1 (fr) * 1999-09-22 2001-03-29 Lts Lohmann Therapie-Systeme Ag Procede et dispositif pour detecter et isoler des composes pharmacologiques contenus dans des melanges de substances

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Publication number Priority date Publication date Assignee Title
EP0707211A1 (fr) * 1994-10-15 1996-04-17 BEHRINGWERKE Aktiengesellschaft Phase solide réutilisable pour les réactions des liaisons spécifiques
US5783455A (en) * 1994-10-15 1998-07-21 Behring Diagnostics Gmbh Regenerable solid phase for carrying out specific binding reactions
WO1999013330A3 (fr) * 1997-09-08 1999-06-17 Roche Diagnostics Gmbh Epuration de substances provenant d'un echantillon biologique
US6635420B1 (en) 1997-09-08 2003-10-21 Roche Diagnostics Gmbh Purification of substances from a biological sample
WO1999036776A1 (fr) * 1997-12-30 1999-07-22 Pharmacia & Upjohn Diagnostics Ab Procede analytique comprenant une addition dans deux ou plusieurs positions et dispositif et trousse d'analyse associes
WO2001022078A1 (fr) * 1999-09-22 2001-03-29 Lts Lohmann Therapie-Systeme Ag Procede et dispositif pour detecter et isoler des composes pharmacologiques contenus dans des melanges de substances
US7232690B1 (en) 1999-09-22 2007-06-19 Lts Lohmann Therapie-Systeme Ag Method and device for detecting and isolating pharmacological compounds being contained in substance mixtures
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