WO1994000763A1 - Procede de determination d'immunocomplexes multiples sur une surfface par spectroscopie - Google Patents

Procede de determination d'immunocomplexes multiples sur une surfface par spectroscopie Download PDF

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Publication number
WO1994000763A1
WO1994000763A1 PCT/US1993/006020 US9306020W WO9400763A1 WO 1994000763 A1 WO1994000763 A1 WO 1994000763A1 US 9306020 W US9306020 W US 9306020W WO 9400763 A1 WO9400763 A1 WO 9400763A1
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WIPO (PCT)
Prior art keywords
light
binding pair
immobilized
reactive surface
sample
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PCT/US1993/006020
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English (en)
Inventor
John David Walsh
Wim J. M. Heuvelsland
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Akzo Nobel N.V.
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Filing date
Publication date
Application filed by Akzo Nobel N.V. filed Critical Akzo Nobel N.V.
Priority to EP93915466A priority Critical patent/EP0647321A4/fr
Priority to JP6502562A priority patent/JPH08500667A/ja
Priority to AU45438/93A priority patent/AU4543893A/en
Publication of WO1994000763A1 publication Critical patent/WO1994000763A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • 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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin
    • 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
    • G01N33/53—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
    • G01N33/553—Metal or metal coated
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex

Definitions

  • the invention relates to a process of detecting one or more analytes in a sample through light-based methodology, especially that of scattered total internal reflectance spectroscopy or reflectance, using a single, multi-functional reactive surface.
  • STIR scattered total internal reflectance
  • This is an analytical method that relies on the differences in the optical properties of two mediums to investigate a sample deposited at, or very near to, the interface of the two mediums.
  • STIR analysis takes advantage of the phenomenon of total internal reflectance wherein light, when directed at or near a critical angle through a high refractive index material toward the interface of that material with a second material having a lower refractive index, is almost completely reflected from that interface except for a microscopic evanescent light wave which propagates into the second material for only a short distance.
  • Patent 3,939,350 when materials that are fluorescently labeled are brought to the interface of materials with different refractive indexes and within the field of the evanescent wave, the molecules can be made to fluoresce. In STIR spectroscopic analysis what is detected is not the fluorescence of a labeled molecule near the interface but rather the elastic scattering of an evanescent light wave by a light-scattering material such as colloidal gold, red blood cells or latex particles that are brought near the interface.
  • a light-scattering material such as colloidal gold, red blood cells or latex particles that are brought near the interface.
  • an "evanescent wave” refers to a non-propagating light wave such as a wave in the region of a surface on the side of the surface opposite the side of illumination, produced when the illuminating light undergoes total internal reflection.
  • light-scattering material means a molecule or a material which causes the incident light to be elastically scattered.
  • the critical angle can only exist in the higher refractive index medium.
  • Total internal reflectance occurs exclusively when an interface between materials of different refractive indexes is illuminated from the higher refractive index medium beyond the critical angle, causing the incident light wave to be reflected unless it is perturbed by diffraction, scatter or absorption.
  • the angle at which incident light is most suitable for STIR spectroscopic analysis of a material deposited on the interface of two materials is, in practice, slightly greater than the theoretical angle calculated as above.
  • the angle at which incident light is suitable can be less than and equal to ⁇ c, which angles do produce evanescent wave, and also provide a useful signal.
  • results of separate tests for an individual sample would be unambiguously determined by use of a multi-analyte test performed on a single, multi-functional reactive surface.
  • routine blood typing requires testing for both ABO and Rh antigens, and the correct identification of a blood type with a given sample is ensured in a single reactive surface test, whereas in a multi-cuvette test there always remains the possibility that the samples tested in the different cuvettes are not of the same origin.
  • controls were performed in either the same or a separate cuvette.
  • the control is a competing pre-reaction, which may lower the sensitivity of the test by raising background levels of scattering.
  • Such a probability of poor controls would also be greatly reduced in a single reactive surface, multi-functional test.
  • Still yet another advantage of a single reactive surface, multi-functional test would be the economic benefits afforded by such an assay. For example, many different fluid samples could be assayed for the presence or absence of different analytes at the same time. This is in contrast to currently available methods wherein each test requires use of a separate reactive surface (e.g., cuvette).
  • a separate reactive surface e.g., cuvette.
  • processing or screening a large number of samples is routine, as again for example in blood screening
  • the decrease in the time it takes to assay multiple samples for multiple binding pair members will result in a decrease in the overall cost of performing the assays.
  • such a lower per-as ⁇ ay cost allows for more assays to be performed per unit time, thereby allowing large numbers of assay results to become available more quickly than was previously possible.
  • a still further economic benefit of a single reactive surface, multi-functional test is that less cuvettes would be needed per test, thereby reducing the cost of materials required.
  • the present invention provides a method wherein multiple tests can be performed on a single reactive surface using spectrophotometric or light-based methodology.
  • a multiplicity of binding pair members that are either the same or different are first immobilized in more than one location, such that they are spatially separated, on a single reactive surface of a solid support, such as a cuvette made from an optically transparent material. These spatially separated, immobilized materials are then contacted with a sample which is to be tested for the presence of one or more analytes.
  • At least one additional binding pair member which is labeled with a light-scattering material, is brought into contact with the sample under conditions wherein either the first immobilized member, the analyte and the light-scattering, labeled member or the first immobilized binding pair member and the light-scattering, labeled member form a specifically bound, light-scattering immunocomplex on or near the reactive surface.
  • a light scattering labeled member may bind to the analyte of interest in the sample or to the first immobilized member, but not both. This labeled member will be either a single gold conjugate (eg. anti- IgG gold) or, more probably, a mixture of differently labeled gold conjugates.
  • the reactive surface is next illuminated by an evanescent light wave resulting from total internal reflectance of a light beam incident upon the optically transparent material comprising the reactive surface and is sufficiently narrow to permit individual discrimination between each of the spatially separated, light-scattering complexes in the vicinity of the reactive surface.
  • Elastically scattered light derived from the interaction of the evanescent wave with the spatially separated, light-scattering complexes, is then detected and correlated with the region of the reactive surface from which the evanescent light wave was scattered. The detection may also be made as a function of time of the reaction on the reactive surface.
  • binding pair members may be labeled with fluorescent, chemiluminescent or other compounds made active through light interactions.
  • the invention provides for spectroscopic detection of multiple variants of a single analyte, simultaneous detection of different analytes, and/or quality control features, where in each case only a single, multi-functional reactive surface is used.
  • Figure 1 is a cross-sectional view of a cuvette which may be used to practice the method of detecting binding pair members in accordance with the invention.
  • Figure 2 is a diagram depicting spatially separated binding pair members immobilized on a reactive surface of an optically transparent material such as the cuvette in Figure l, and a path of a continuously scanned light wave incident upon the optically transparent material for practicing the method according to the invention.
  • Figure 9 depicts the set-up of a reflectometer measuring the thickness of a flat carrier's coating.
  • Figure 10 depicts the interaction of antibodies spotted and adsorbed onto wafers with various antigens.
  • Figure 11 is a graph showing the interactions of three different antigens with three antibodies adsorbed onto one piece of wafer.
  • the present invention provides a method that solves the above-described problems currently associated with conventional spectroscopic and light-based analysis, and in particular, STIR analysis, by providing an assay method wherein multiple tests can be performed on a single reactive surface.
  • the presence of more that one analyte in a sample 20 can be detected on a single reactive surface 22 of a cuvette 11.
  • the structure and the geometry of the reactive surface is not limited to the cuvette described above. However, from this point on, the term "cuvette" will be used to refer to any reactive surface regardless of geometry and structure. Controls may also be detected on the same reactive surface 22 as that used for detecting the presence of a binding pair member in sample 20.
  • STIR-based detection method of the present invention utilizes measurement of elastically scattered light derived from a scanning evanescent wave which is disturbed by the presence of a light-scattering material 15 brought near the interface of reactive surface 22 upon which an i munochemical reaction is taking, or has taken, place.
  • a light-scattering material 15 brought near the interface of reactive surface 22 upon which an i munochemical reaction is taking, or has taken, place.
  • the measurement of reflected light from multiple test loci will detect immunocomplexes formed.
  • binding pair member or “member” are meant to designate materials which are capable of specifically interacting with one another so as to form a stable immunocomplex.
  • typical binding pair members include analytes, antibodies, antigens, haptens, intact cells containing antigenic determinants, proteins, polypeptides, nucleic acids, chelators, both DNA-binding and immunoglobulin-binding proteins, biological receptors, lipids, lectins, carbohydrate residues, streptavidin and biotin- derivatized macromolecules.
  • a suitable apparatus and reactive surface for the STIR spectroscopic detection of the present invention is the same as that disclosed in U.S. Patent No. 5,017,009, while another suitable reactive surface is that provided by the cuvette disclosed in U.S. Patent No. 4,979,821.
  • Cuvettes are comprised of suitable optically transparent materials such as glass, quartz, silicon, plastics such as polycarbonate, acrylic, or polystyrene, or oils comprising silicone or high molecular weight hydrocarbons. Preferred in the method of the invention is the use of transparent polycarbonate plastic cuvettes, as these are economical and yet durable.
  • binding pair members 12 such as, preferably antibodies or molecules carrying antigenic determinants, are first immobilized, in a spatially separated way, onto a reactive surface of an optically transparent material, for example, the inner wall of a cuvette.
  • control binding pair members are also placed upon the same reactive surface.
  • the binding pair members may be immobilized on the reactive surface of the optically transparent material in any convenient pattern or shape in the practice of the invention. However, there must be sufficient separation of the immobilized binding pair members that the reaction of each of these members individually with the corresponding binding pair member in a fluid sample and/or a light-scattering labeled member can be measurably and discriminately detected without substantial interference due to nearby immobilized binding pair members. It is preferred that the binding pair members be applied in the shape of either a circle or a rectangular stripe. The first is most conveniently accomplished by applying a small droplet of binding pair member onto the reactive surface and allowing the droplet to dry.
  • micropipets or icrocapillary tubes may be conveniently used for spotting members onto the reactive surface. It is also preferred to use mechanical means for the application of these members to the reactive surface, since such a method is convenient, reproducible and cost-saving.
  • the advantages of mechanization make mechanical application of a binding pair member particularly desirable when the STIR assay is used in large-scale testing, such as routine screening applications. Examples of this use can be seen in infectious disease testing or typing of donated blood.
  • Mechanical application methods include those such as used in ink jet spraying similar to those commonly used in electronic printers or touch pad strips. For more specialized or individualized tests, manual application may be preferred.
  • the binding pair members may first be put into a solution to facilitate the process of depositing these samples onto the reactive surface.
  • Suitable solutions for this purpose have only the general requirement that their use not substantially interfere with any subsequent specific binding reaction between binding pair members in the sample, on the reactive surface and a light-scattering, labeled member 15. Again, when light-scattering is not a part of the spectroscopic methodology, a labeled member may not be needed.
  • Suitable solutions include aqueous and organic solutions as required by the particular binding pair member that is to be deposited.
  • Such solutions may also be buffered with, for example, phosphate, TRIZMA (Sigma) or HEPES (Sigma) and preferably, carbonate-bicarbonate buffer, pH 9.5, or borate buffers at pH 9.3. It is preferred in the process of the invention that either carbonate or borate buffer be employed when necessary for the application of a protein binding pair member onto the reactive surface.
  • Binding pair members can also be attached to the reactive surface by covalent forces.
  • the reactive surface may be derivatized directly with a variety of chemically reactive groups which then, under certain conditions, form stable covalent bonds with the applied binding pair member.
  • the reactive surface may first be coated with chemically-derivatized polymers, such as dextran or PEG, which then bind applied binding pair members covalently.
  • Certain types of detergents may also be coated to the reactive surface, then derivatized in situ, and reacted with binding pair members.
  • the reactive surface may be coated with streptavidin through physical adsorption, then reacted with a biotin-labeled binding pair member to create a well characterized, biologically responsive surface.
  • the member After the binding pair member has been deposited on spatially separated areas of the reactive surface, the member is then allowed to dry and thereby become immobilized on the reactive surface. Evaporation is the preferred drying method, and may be performed at room temperature (about 25°C) . When desired, the evaporation may be performed at elevated temperatures, so long as that temperature does not sufficiently inhibit the ability of the immobilized members to specifically interact with their corresponding binding pair members or light-scattering labeled member. For example, where the immobilized binding pair member is a protein, non-denaturing temperatures should be employed.
  • the reactive surface is preferably treated so as to block non-specific interactions between the immobilized member and corresponding members in a fluid sample which is to be tested.
  • one blocking material is a protein. Others can be detergents and long-chain water soluble polymers. Most preferred in this instance is bovine serum albumin (BSA) , due to its availability and low cost.
  • BSA bovine serum albumin
  • the blocking material may be conveniently applied to the reactive surface as an aqueous or buffered aqueous solution.
  • the blocking solution may be applied to the reactive surface at any time after the first binding pair members are immobilized. It is preferred that cuvettes be blocked as soon as is practical after immobilization. A sample to be tested for the presence of corresponding binding pair members is then brought into contact with the immobilized members on the reactive surface. The only general requirement of this process step is that the sample be in direct contact with the spatially separated immobilized binding pair members. Mild mixing of the fluid sample after bringing it in contact with the immobilized member is also preferred in the process of the present invention, but is not required. Such mixing may help to ensure close contact between the fluid sample and the immobilized binding pair member.
  • a second binding pair member that specifically binds to either the immobilized member or its corresponding binding pair member in the solution is labeled with a light-scattering material such as colloidal gold, red blood cells or latex particles.
  • the light-scattering labeled member in the method of the invention is chosen such that it specifically interacts with either the immobilized member on the reactive surface or with one or more corresponding binding pair members possibly present in the solution, thereby forming spatially separated light-scattering complexes with these materials.
  • the light-scattering, labeled member may be included in the sample prior to contacting the fluid sample to the immobilized member or may be added to the sample after the latter is brought into contact with the immobilized member.
  • the fluid sample and the light- scattering labeled member may be brought into contact with the immobilized member simultaneously.
  • a light wave 3 is directed at the optically transparent material comprising the reactive surface, which illuminates the region of the reactive surface to which an individual light-scattering immunocomplex is located.
  • Suitable light sources for generating the incident light beam 3 in the method of the invention provide collimated or uncollimated light, polarized or unpolarized light, or monochromatic or polychromatic light.
  • Preferred light sources include lasers, light emitting diodes, flash lamps, arc lamps, incandescent lamps and fluorescent discharge lamps.
  • the light source used to illuminate the reactive surface is a low wattage helium-neon laser.
  • sources of light such as ultra-violet, visible, and near-IR may also be used in the proper setting.
  • the incident light wave that is internally reflected is preferably continuously scanned across the optically transparent material such that an evanescent wave derived therefrom will interact with only a small number of light-scattering complexes on or near the reactive surface at any given time.
  • continuous scanning of the incident light wave is not required in the process of the present invention as the incident light wave may be sequentially directed only at those areas of the reactive surface which will give rise to an evanescent wave in the area of the reactive surface on which materials have been immobilized.
  • the incident light wave is scanned across the transparent material and any scattered light derived from the evanescent wave is continuously monitored and correlated with the position of the optically transparent material being illuminated and as a function of time.
  • the scan rate of incident light wave across the optically transparent material is not particularly critical in the practice of the invention, with the only general requirement that the rate not be so fast that correlation of the scattered light detected with the position of the transparent material being illuminated is not impaired.
  • the transparent material may be repeatedly illuminated in the same manner described above, such that the intensity of scattering can be determined as a function of time.
  • the reactive surface were coated with a negative control, positive control and binding pair member, each within distinct spatially separated regions, then the reaction on the reactive surface between the immobilized binding pair members, the binding pair members in the sample, and/or the light-scattering binding pair members could be measured after a suitable incubation period with only a single scattered detection, i.e., scan.
  • the number of scan cycles can vary as dictated by the assay. It should also be recognized that the number of scans required is independent of the scanning rate of the incident light wave.
  • multiple scans be used when the assay is intended to be quantitative, since the time-dependency (i.e. rate) of the increase or decrease in the amount of light scattered may be more accurately indicative of the levels of the binding pair members present in the fluid sample than the total amount of scatter by the reaction at any given reaction point in time. Additionally, the use of multiple scans can provide a data set over which the increase in scattered light detected is of a known function with respect to time. Measuring the rate of change of the intensity of scattered light from a given region of the reactive surface versus time provides a reaction rate. By using reaction kinetics, the rate is correlated to a quantitative measure of analyte concentration in the sample solution.
  • Another method is to integrate the scattered light intensity versus time.
  • the area obtained by this integration is correlatable to the concentration of the analyte in solution.
  • Suitable photodetection means in the process of the present invention include photon detectors such as photomultiplier tubes, photodiodes such as PIN diodes or gallium- aluminum-arsenide diodes, or cadmium sulfide photoresistive cells, phototubes and pyrolytic detectors. Preferred is use of a photodiode, such as a Hamamatsu No. G1742 photodiode, in the STIR method of the invention.
  • the detection means are positioned relative to the light source such that the scattered light detected is back-scattered light. It is further preferable that the detection means detect elastically back-scattered light.
  • the binding pair members employed are antigens or antigenic determinants such as antibody- binding epitopes and antibodies or antibody fragments to these determinants. These types of binding pair members are preferred due to both the high specificity and selectivity of the antibody-antigen reaction as well as the great utility of such a test in the determination of the components of, in particular, blood or serum. It must be recognized, however, that the process of the invention is not meant to be limited to only such interactions, as the method disclosed herein is equally applicable and is highly useful to the detection of, for example, specific DNA or RNA analytes in a sample.
  • the process of the invention provides detection of the specific interaction between proteins and nucleic acids or lipids, immunoglobulins and immunoglobulin binding proteins such as protein A and protein G, biological receptor molecules and their corresponding ligands, lectins and specific carbohydrate moieties, and streptavidin and biotin-labeled macromolecules.
  • proteins and nucleic acids or lipids immunoglobulins and immunoglobulin binding proteins such as protein A and protein G, biological receptor molecules and their corresponding ligands, lectins and specific carbohydrate moieties, and streptavidin and biotin-labeled macromolecules.
  • these additional embodiments of the present invention may be either direct reactions or may be based upon competitive reactions.
  • the reactions that take place on the reactive surface may be of several types, the following of which are not meant to be an exhaustive list but rather merely exemplary of suitable techniques.
  • antibodies to different antigens are individually deposited onto the reactive surface.
  • the analytes to be detected in the fluid sample in this embodiment are the antigens to which the immobilized antibodies specifically bind.
  • the light-scattering, labeled member of this embodiment may then be a second antibody which has been labeled with a light-scattering material and binds the same or a different antigenic determinant than the immobilized member in a non-competitive way.
  • the specific analyte When the specific analyte is present in the fluid sample, a reaction occurs on the reactive surface between the antibody coated thereon and the antigen in the fluid sample. A second reaction takes place simultaneously between the antigen in the fluid sample and the labeled antibody, resulting in the formation of an antibody-antigen-labeled antibody immunocomplex on or near the reactive surface.
  • the amount of an evanescent light wave scattered by the light-scattering complex provides an accurate indication of the presence of the complex formed on or near the reactive surface and, thereby, the presence of the analyte in the fluid solution.
  • the amount of scattered light detected provides a quantitative analysis of the concentration of the analyte in the fluid solution.
  • both the immobilized member and the light-scattering labeled member are the same antigen or series of antigens or antigenic determinants.
  • the analytes to be detected in the fluid sample in this embodiment are antibodies which bind specifically to both the immobilized member and the light-scattering labeled member, forming a specific link between the two on or very near the reactive surface. This embodiment is demonstrated in the examples.
  • the immobilized binding pair member is a material with an antigenic determinant, while the assay tests for the presence of a specific antibody to that determinant in the sample.
  • the labeled, light-scattering member is an antibody specific for the antigenic determinant binding pair member.
  • the sample is contacted with the immobilized member such that an antigen-antibody reaction is allowed to occur.
  • the light-scattering, labeled antibody is then introduced into the fluid sample and the amount of labeled antibody forming an immunocomplex with the antigen on the reactive surface is determined by the amount of elastically-scattered light derived from an evanescent light wave.
  • this embodiment of the instant invention also provides for a quantitative analysis of the amount of analyte in the fluid sample.
  • an optically transparent cuvette 11 providing a reactive surface 22 with immobilized member 12 deposited thereon is depicted.
  • An incident light beam 3 is directed to the optically transparent material at an angle that produces an evanescent lightwave which illuminates complexes on or near the reactive surface.
  • the scattered light derived from an evanescent light wave is detected in the process of the invention and correlated with the region of the reactive surface which generated the scattered light.
  • the placement of the immobilized binding pair members 12 on the reactive surface 22 and with respect to the area of illumination of the optically transparent material that comprises the reactive surface 22 is schematically depicted.
  • the relation of the deposited material to a scan (arrow) of the incident light wave 3 (stippled area), is also schematically depicted.
  • the evanescent wave derived from the scanned incident light wave 3 moves across the reactive surface 22, the scattered light generated by any elastically scattered light derived from individual light-scattering complexes located at or near the reactive surface 22 are individually detected.
  • the region of the reactive surface 22 that does not have an immobilized member 12 placed thereon serves to both differentiate regions of the reactive surface 22 whereon member 12 has been immobilized from those on which no material resides, and also provides background light scatter data over which any specific light-scattering reaction can be discriminated.
  • the relative width of the incident internally reflected light wave 3 at the reactive surface as compared to the immobilized binding pair member 12 is also schematically depicted.
  • the immobilized binding members are either the human immunodeficiency virus (HIV-1) 160 kD glycoprotein (gpl60; Advanced Biosciences Laboratories, Inc., Kensington, MD) or the hepatitis B virus surface antigen (HBsAg; BioTech Resources Inc., San Antonio, TX) .
  • the light-scattering binding pair members are the above- mentioned proteins labeled with colloidal gold of approximately 70nm in diameter, prepared as described by Turkevich et al. (J. Phy ⁇ . Chem. 57:670-673, 1953) , and incorporated herein by reference.
  • the positive control binding pair member was the ⁇ -HBsAg monoclonal antibody 233D (Biotechnology Research Institute, Rockville, MD) .
  • Multi-analyte cuvettes were prepared by placing one 1.0 microliter droplet each of gpl60 (2 ⁇ g/ml) and HBsAg (l ⁇ g/ l) about 1 millimeter apart on the reactive surface of the cuvette in a horizontal plane along the incident light wave path, as depicted in Figure 2.
  • a cuvette of the structure described in U.S. Patent No. 4,979,821 was used. These deposited materials were then immobilized onto the cuvette's reactive surface by air drying overnight at room temperature. The reactive surface was blocked by filling the cuvette with a solution of 2% BSA in 25mM Hepes/Tris, pH 7.4 for 60 minutes at room temperature. The blocking solution was aspirated and the cuvettes washed once with the same buffer, then air dried and stored at room temperature in an atmosphere of low humidity.
  • the cuvettes were placed into a circular carousel and illuminated with a rotating laser beam, as depicted in Figure 1.
  • the test human serum sample was added to the cuvette in sufficient volume to cover the region of the reactive surface to which the two binding pair members had been immobilized.
  • a solution containing assay buffer and a 50:50 mixture of gpl60-gold sol conjugate and HBsAg-gold sol conjugate was added.
  • the contents of the cuvette were mixed, then the amount of light- scattering was recorded continuously for 10 minutes.
  • the data is presented as a series of plots representing an increase in light-scattering (y-axis) due to the presence of gold-labeled immunocomplexe ⁇ accumulating on or above distinct regions of the cuvette's reactive surface (x-axis) . These complexes are detected by a laser beam scanning across the cuvette. Due to this fact, the 0-50% region of the x- axis was coated with gpl60 while the 50-100% region was coated with HBsAg, as described above. The plots depict light scattering readings every two minutes up to ten minutes, with the lowest line representing the first reading taken approximately ten seconds after the addition of the mixed gold sol conjugate.
  • Figures 3-6 demonstrate the results obtained with four different human serum samples, containing antibodies to gpl60, antibodies to HBsAg, antibodies to both or to neither.
  • Figure 3 shows the response obtained when normal human serum was added to the cuvette. This serum contains no antibodies to either gpl60 or HBsAg and thus does not generate a light scattering signal in any region of the cuvette.
  • the sample applied to the cuvette was human serum containing antibodies to gpl60, but none to HBsAg.
  • the response demonstrated a specific, time- dependent increase in scattered light from the region of the cuvette corresponding to the position where gpl60 was immobilized. No response was detected within the HBsAg coated region.
  • the sample applied to the cuvette was human serum containing antibodies to HBsAg, but none to gpl60.
  • the results obtained were the inverse of those in Figure 4.
  • a specific, time-dependent increase in scattered light was found in the region of the cuvette corresponding to the position where HBsAg was immobilized, but not where gpl60 was immobilized.
  • EXAMPLE 2 The multi-functional cuvettes used in this example were prepared in a slightly different manner to those described in Example 1. Thin stripes of HBsAg (2.5 ⁇ g/ml) and 233D (lO ⁇ g/ml) were immobilized onto the reactive surface of the cuvette on a plane perpendicular to and bisecting the scanning incident light beam. The stripes were applied using modified single well sample applicators from Pharmacia's PhastSystem R (Pharmacia, Uppsala, Sweden) . These deposited binding pair members were allowed to air dry at room temperature onto the cuvette's reactive surface. The cuvettes were blocked, washed and stored as described in Example 1.
  • the assay conditions were identical to those described in Example 1, except that only a single gold conjugate (HBsAg coated colloidal gold) was added to the cuvette containing the test serum sample.
  • the aim of these experiments was to demonstrate the feasibility of incorporating a reagent positive control into each cuvette.
  • the 0-50% region of the x-axis of the cuvette was coated with HBsAg while the 50-100% region was coated with 233D monoclonal antibody. This antibody will react directly with the HBsAg-gold conjugate generating a strong light- scattering signal with every cuvette, regardless of the presence of specific analyte or not.
  • Figure 7 demonstrates that when a serum sample negative for a specific analyte (in this case anti-HBsAg antibody) was applied to the cuvette, there was no time- dependent increase in scattered light from the HBsAg coated region of the reactive surface. However, there was a strong reaction in the region of the reactive surface coated with 233D antibody, as the HBsAg-gold conjugate bound directly and specifically to this antibody. This reaction can be quantitated and used to assess the reagents integrity.
  • a specific analyte in this case anti-HBsAg antibody
  • the human serum sample applied to the cuvette contained antibodies to HBsAg.
  • a specific, time-dependent increase in scattered light was observed in the region of the cuvette where HBsAg was coated.
  • there was a strong reaction in the 233D coated region very similar to that seen in Figure 7, again demonstrating the integrity of the colloidal gold reagent.
  • Figure 9 shows the set-up of the reflectometer.
  • the angle of incidence is the Brewster angle for a silicon/water interface, at which the reflectance of the parallel polarized light is zero. This is used to position the laser and the cuvette, which has windows perpendicular to the incident and the reflected light.
  • EXPERIMENT 3A Single Spot Per Wafer
  • silicon wafers coated with polystyrene as described above were spotted with antibodies against hCG, HBsAg, and rubella.
  • one set of wafers consisted of individual wafers where only one antibody was spotted in one place on each wafer and allowed to adsorb onto the wafer. A different wafer was used for each determination.
  • concentrations of the reagents used are given in Table 2.
  • EXPERIMENT 3B Multiple Spots Per Wafer
  • Another set of wafers consisted of wafers coated as above, where each antibody was spotted onto a single wafer in such a manner that each spot was distinct from the other, and allowed to dry for one day.
  • Antibodies to hCG were placed on the left third of the wafer, antibodies to HBsAg were placed in the center and antibodies to rubella were placed in the remaining third. Then a different antigen was applied to each wafer, and the reflectance measured.
  • the concentrations of the antibodies and antigens used are given in Table 2 above.

Abstract

L'invention concerne un procédé de détection de la présence de plus d'un analyte dans un échantillon fluide en utilisant une seule surface réactive dans un essai spectroscopique.
PCT/US1993/006020 1992-06-24 1993-06-23 Procede de determination d'immunocomplexes multiples sur une surfface par spectroscopie WO1994000763A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP93915466A EP0647321A4 (fr) 1992-06-24 1993-06-23 Procede de determination d'immunocomplexes multiples sur une surfface par spectroscopie.
JP6502562A JPH08500667A (ja) 1992-06-24 1993-06-23 分光学を用いて同一表面上の多数の免疫複合体を測定する方法
AU45438/93A AU4543893A (en) 1992-06-24 1993-06-23 Method for determining multiple immunocomplexes on one surface using spectroscopy

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US90369392A 1992-06-24 1992-06-24
US07/903,693 1992-06-24

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996009532A1 (fr) * 1994-09-22 1996-03-28 Abbott Laboratories Procede utilisant un guide d'ondes optique pour detecter des evenements de liaison specifique par diffusion de la lumiere
EP0851229A1 (fr) * 1996-12-23 1998-07-01 Bayer Corporation Utilisation des cristaux liquides thermochromiques dans des procédés de diagnostiques basés sur la réflectométrie
US6231733B1 (en) 1993-04-17 2001-05-15 Kurt Nilsson Immobilized carbohydrate biosensor
WO2006032407A1 (fr) * 2004-09-21 2006-03-30 Universita' Degli Studi Di Milano Procede pour mesurer des interactions moleculaires par diffusion de la lumiere laser (lls)
US7944563B2 (en) 2007-10-22 2011-05-17 Fujifilm Corporation Sensing apparatus

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WO1984002578A1 (fr) * 1982-12-21 1984-07-05 Comtech Res Unit Technique d'analyse
US4908508A (en) * 1987-02-12 1990-03-13 Akzo N.V. Process and apparatus for determining thicknesses of layers
US5017009A (en) * 1986-06-26 1991-05-21 Ortho Diagnostic Systems, Inc. Scattered total internal reflectance immunoassay system

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WO1984002578A1 (fr) * 1982-12-21 1984-07-05 Comtech Res Unit Technique d'analyse
US5017009A (en) * 1986-06-26 1991-05-21 Ortho Diagnostic Systems, Inc. Scattered total internal reflectance immunoassay system
US4908508A (en) * 1987-02-12 1990-03-13 Akzo N.V. Process and apparatus for determining thicknesses of layers

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6231733B1 (en) 1993-04-17 2001-05-15 Kurt Nilsson Immobilized carbohydrate biosensor
US6887689B2 (en) 1993-04-19 2005-05-03 Kurt Nilsson Immobilized carbohydrate biosensor
US7244582B1 (en) 1993-04-19 2007-07-17 Kurt Nilsson Immobilized carbohydrate biosensor
US7625722B2 (en) 1993-04-19 2009-12-01 Kurt Nilsson Immobilized carbohydrate biosensor
WO1996009532A1 (fr) * 1994-09-22 1996-03-28 Abbott Laboratories Procede utilisant un guide d'ondes optique pour detecter des evenements de liaison specifique par diffusion de la lumiere
US5599668A (en) * 1994-09-22 1997-02-04 Abbott Laboratories Light scattering optical waveguide method for detecting specific binding events
US5843651A (en) * 1994-09-22 1998-12-01 Abbott Laboratories Light scattering optical waveguide method for detecting specific binding events
EP1441217A2 (fr) 1994-09-22 2004-07-28 Abbott Laboratories Procédé utilisant un guide d'ondes optique pour détecter des événements de liaison spécifique par diffusion de la lumière
EP1441217A3 (fr) * 1994-09-22 2007-07-04 Abbott Laboratories Procédé utilisant un guide d'ondes optique pour détecter des événements de liaison spécifique par diffusion de la lumière
EP0851229A1 (fr) * 1996-12-23 1998-07-01 Bayer Corporation Utilisation des cristaux liquides thermochromiques dans des procédés de diagnostiques basés sur la réflectométrie
WO2006032407A1 (fr) * 2004-09-21 2006-03-30 Universita' Degli Studi Di Milano Procede pour mesurer des interactions moleculaires par diffusion de la lumiere laser (lls)
US7944563B2 (en) 2007-10-22 2011-05-17 Fujifilm Corporation Sensing apparatus

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EP0647321A4 (fr) 1997-10-22
EP0647321A1 (fr) 1995-04-12
CA2137259A1 (fr) 1994-01-06
JPH08500667A (ja) 1996-01-23
AU4543893A (en) 1994-01-24

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