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
• • 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
  • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
  • G01N33/567—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds utilising isolate of tissue or organ as binding agent
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in epitope analysis
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/805—Optical property

Definitions

• the invention relates to the field of biosensors and methods comprising detecting antigens that specifically bind to an antibody, antibody fragment, or phage.
• a typical pipeline for identifying potential therapeutic and diagnostic antibodies includes: (1) phage display and phage panning experiments on soluble protein or cellular associated proteins (in the soluble form or expressed on cells); (2) a phage ELISA performed on soluble protein (for cellular targets - a peptide or protein-mimic of the cellular associated protein); (3) the display gene in the phage genome is subcloned via molecular biology techniques to a soluble antibody fragment expressing plasmid; (4) The antibody fragment then is expressed and purified; (5) once purified the antibody fragment can be tested for cellular functional binding in ELISA, FACS, Guava or FMAT; (6) The lead antibody fragment is analyzed for binding kinetics; and (7) the top antibody lead is then cloned into a full antibody expression vector for large scale production, kinetic analysis and in vivo efficacy models.
• Typical assays for analysis of functional binding of phage to protein targets associated with cells include whole mammalian or bacterial cell enzyme-linked immunosorbent assay (ELISA), flow cytometry (Fluorescence Activated Cell Sorter, FACS), Guava microcytometry products (Guava Technologies, Hayward, CA), and fluorescence microassay technology (FMAT).
• ELISAs have high background binding of phage, because cells are complex and phages have a tendency to bind non-specif ⁇ cally. Background binding in ELISA is intensified due to amplification of the binding signal.
• Cellular ELISAs are also difficult due to the need of many washes between steps, which is cumbersome if the cells are non-adherent as a centrifugal spin is required between each wash. Often adherent cells must be fixed in order to keep the cells attached to the ELISA plate during washes, either manually or on a plate washer. The fixation can change the natural epitopes of the protein on cells. Phage binding in FACs and Guava is also difficult, because each phage clone needs to be purified to get enough phage for a signal.
• One embodiment of the invention provides a method of detecting binding of a binding partner to a phage.
• the method comprises immobilizing a crude phage preparation, unconcentrated phage preparation, non-homogenous phage preparation, or a combination thereof on a biosensor and contacting the biosensor with the binding partner. Binding of the binding partner to a phage immobilized on the biosensor is detected.
• the binding partner can be a small molecule, a carbohydrate, a polymer, a peptide, a soluble protein, a cellular receptor, an antigen mimic of a cellular receptor, a cell, a mammalian cell, or a mammalian cell surface protein.
• the phage preparation and antigen do not necessarily comprise a detectable label.
• the phage preparation can be a phage display library.
• the phage preparation can be passively immobilized to the biosensor or can be immobilized to the biosensor by an antibody specific for a phage coat protein.
• the antibody or antibody fragment can be immobilized to the biosensor by binding to a protein that is bound to the biosensor. If the antibody or antibody fragment comprises a tag, the antibody or antibody fragment can be immobilized to the biosensor by antibodies specific for the tag.
• the biosensor can be a colorimetric resonant reflectance biosensor or an evanescent wave-based biosensor. Another embodiment of the invention provides a method for determining epitope classes of antibodies in an antibody population.
• the method comprises immobilizing a display phage, antibody, or antibody fragment to a biosensor and contacting the biosensor with a binding partner that specifically binds to the display phage, antibody, or antibody fragment immobilized to the biosensor, under conditions suitable for binding of the binding partner to the display phage, antibody, or antibody fragment.
• the antibody population is contacted with the biosensor.
• a detectable signal generated by binding of the antibody population to the binding partner indicates that different epitope classes are present in the antibody population than in the immobilized display phage, antibody, or antibody fragment.
• the antibody population, binding partner and immobilized display phage, immobilized antibody, or immobilized antibody fragment do not necessary comprise a detectable label.
• the antibody population can comprise phage clones, antibody fragments, full antibodies, phage displaying a full antibody, phage displaying an antibody fragment, antibodies from a hybridoma, and antibodies from a phage display screen.
• the display phage can be a purified phage preparation, a crude phage preparation, an unconcentrated phage preparation, or a non-homologous phage preparation.
• the binding partner can be a small molecule, a carbohydrate, a polymer, a peptide, a soluble protein, a cellular receptor, an antigen mimic of a cellular receptor, a mammalian cell, or a mammalian cell surface protein.
• the mammalian cell surface protein can be a membrane-associated protein, a single transmembrane protein, a multi- transmembrane protein, or a protein channel.
• the biosensor can be a colorimetric resonant reflectance biosensor or an evanescent wave-based biosensor.
• the invention provides methods to, e.g., resolve low concentrations of binding partners, rank protein affinities, work with samples comprising complex mixtures, and perform off-rate ranking analysis.
• Figure IA-C depicts an antibody and antibody fragments, F(ab) and scFv.
• Figure IA shows a full IgG antibody and domains of the IgG.
• Figure IB shows F(ab).
• Figure 1C shows scFv.
• Figure 2 shows titration of bacterial viruses on GA3 BIND® Biosensor
• Figure 3A-B shows F(ab) capture from periplasmic extract on a TIO BIND® Biosensor.
• Figure 3A shows creation of the sF(ab) specific capture surface and the capture of sF(ab).
• Figure 3B shows a graphical representation of the capture of sFab recorded in Figure 3A.
• Figure 4A-D shows scFv capture from periplasmic extract on a TIO BIND® Biosensor.
• Figure 5A shows the creation of scFv specific capture surface and capture of scFv containing a 6xHis and c-myc tag.
• Figure 4B shows capture of purified scFv spiked into PBS and periplasmic extract.
• Figure 4C shows a graphical representation of capture of scFv recorded in Figure 4B.
• Figure 4D shows graphical representation of capture of scFv recorded in Figure 4B.
• Figure 5A-C shows scFv capture from periplasmic extract on a SAl BIND®
• FIG. 5A shows creation of the specific capture surface for proteins expressing a 6x his tag.
• Figure 5B shows capture of scFv spiked into PBS and periplasmic culture.
• Figure 5C shows graphical representation of scFv capture of purified scFv spiked into PBS and periplasmic extract.
• Figure 6A-C shows scFv capture from periplasmic extract on a GAl BIND®
• FIG. 6 A shows creation of specifc capture surface for proteins containing a c-myc tag.
• Figure 6B shows capture of purified scFv spiked into PBS and a periplasmic extract.
• Figure 6C shows a graphical representation of scFv capture from PBS and periplasmic extract.
• Figure 7A-E shows capture of IgGS from hybridoma supernatants using an anti-
• Figure 7A shows creation of specific mouse IgG capture surface.
• Figure 7B shows creation of specific mouse IgG capture surface.
• Figure 7C shows capture of IgGs from hybridoma media.
• Figure 7D shows a graphical representation of antigen, parental cell line and antigen expressing cell line binding to the
• Figure 7E shows a tabular representation of antigen, parental cell line and antigen expressing cell line binding to the IgGs captured on the biosensor surface with the normalization of this binding to the amount of IgG captured on the biosensor surface.
• Figure 8A-E shows IgG capture from serum using an anti-Fc TIO BIND® Biosensor.
• Figure 8 A shows creation of a specific mouse IgG binding surface.
• Figure 8B shows capture of IgGs from serum.
• Figure 8C shows capture of IgGs from serum.
• Figure 8D shows a graphical representation of the capture of sFab recorded in Figure 8B.
• Figure 8E show a graphical representation of the capture of sFab recorded in Figure 8C.
• Figure 9A-C shows a drug-anti-drug assay in serum using a GAl BIND®
• FIG. 9A shows creation of a surface for the capture of an IgG, an anti-drug.
• Figure 9B shows capture of the anti-drug from PBS, 11% and 30% serum on the 20 ug/ml drug surface.
• Figure 9C shows capture of the anti-drug from PBS, 11% and 30% serum on the 0 ug/ml drug surface.
• Figure lOA-C shows IgG capture from serum using an anti-Fc TIO BIND®
• Figure 1OA shows creation of the drug surface for the capture of anti-drug.
• Figure 1OB shows capture of the anti-drug from PBS, 11% and 30% serum on the 50 ug/ml Drug surface.
• Figure 1OC shows capture of the anti-drug from PBS, 11% and 30% serum on the 0 ug/ml drug surface.
• Figure 1 IA-D shows endpoint analysis of antibody binning and identification of sandwich pairs of antibodies on an anti-FC TIO BIND® Biosensor.
• Figure 1 IA shows creation of mouse IgG specific surface.
• Figure 1 IB shows capture of layer 1 (Antibody).
• Figure HC shows capture of layer 2 (Antigen by Antibody).
• Figure HD shows capture of layer 3 (Antibody by Antibody- Antigen Complex).
• the methods of the invention comprise the use of a biosensor that can be used to, inter alia, detect inorganic or organic material, such as protein, DNA 3 small molecules, viruses, cells, and bacteria, without the requirement of a detectable label, such as fluorescent or radioactive labels.
• a biosensor that can be used to, inter alia, detect inorganic or organic material, such as protein, DNA 3 small molecules, viruses, cells, and bacteria, without the requirement of a detectable label, such as fluorescent or radioactive labels.
• biosensors can be used in the methods of invention, including, but not limited to, photonic crystal biosensors (e.g., colorimetric resonant reflectance biosensors, silver nanoparticle array biosensors), interferometric biosensors (e.g., RIfS, dual polarization interferometer, Hartman Interferometer), MEMS biosensors (e.g., cantilevers, resonant membranes), acoustic biosensors (e.g., quartz resonator), microwave biosensors (e.g., dielectric spectroscopy), surface plasmon resonance (SPR) biosensors (e.g., kreitchman SPR biosensors, imaging SPR biosensors, grating coupled imaging SPR biosensors), waveguide biosensors (e.g., input grating coupler biosensors, chirped waveguide graiing biosensors), evanescent wave-based biosensors and any biosensors incorporating an optical waveguide, as described for example in U
• the methods of the invention have utility in, inter alia, the fields of pharmaceutical research ⁇ e.g., primary screening, high throughput screening, secondary screening, quality control, cytotoxicity, clinical trial evaluation), life science research ⁇ e.g., proteomics, protein interaction analysis, DNA-protein interaction analysis, enzyme- substrate interaction analysis, cell-protein interaction analysis), diagnostic tests ⁇ e.g., protein presence, cell identification), and environmental detection (bacterial and spore detection and identification).
• pharmaceutical research e.g., primary screening, high throughput screening, secondary screening, quality control, cytotoxicity, clinical trial evaluation
• life science research e.g., proteomics, protein interaction analysis, DNA-protein interaction analysis, enzyme- substrate interaction analysis, cell-protein interaction analysis
• diagnostic tests e.g., protein presence, cell identification
• environmental detection bacterial and spore detection and identification
• Colorimetric resonant reflectance biosensors comprise a subwavelength structured surface.
• Subwavelength structured surfaces are a type of diffractive optic that can mimic the effect of thin-film coatings. See, e.g., Peng & Morris, "Resonant scattering from two- dimensional gratings," J. Opt. Soc. Am. A, Vol. 13, No. 5, p. 993, May 1996; Magnusson, & Wang, “New principle for optical filters,” Appl. Phys. Lett., 61, No. 9, p. 1022, August, 1992; Peng & Morris, “Experimental demonstration of resonant anomalies in diffraction from two-dimensional gratings,” Optics Letters, Vol. 21, No. 8, p.
• a grating of a photonic crystal biosensor of the invention has a grating period that is small compared to the wavelength of incident light such that no diffractive orders other than the reflected and transmitted zeroth orders are allowed.
• a photonic crystal biosensor can comprise a grating, which is comprised of or coated with a high dielectric constant dielectric material, sandwiched between a substrate layer and a cover layer that fills the grating grooves. Optionally, a cover layer is not used.
• the grating structure selectively couples light at a narrow band of wavelengths. This highly sensitive coupling condition can produce a resonant grating effect on the reflected radiation spectrum, resulting in a narrow band of reflected or transmitted wavelengths. The depth and period of the grating are less than the wavelength of the resonant grating effect.
• the reflected or transmitted color of a colorimetric resonant reflectance biosensors structure can be modified by the addition of molecules.
• the added molecules increase the optical path length of incident radiation through the biosensor structure, and thus modify the wavelength at which maximum reflectance or transmittance will occur.
• a colorimetric resonant reflectance biosensor When illuminated with white light a colorimetric resonant reflectance biosensor reflects only a single wavelength or a narrow band of wavelengths. When molecules are attached to the surface of the biosensor, the reflected wavelength (color) is shifted due to the change of the optical path of light that is coupled into the grating.
• molecules such as specific binding substances to a biosensor surface
• complementary binding partner molecules can be detected without the use of any kind of detectable label, e.g., a fluorescent probe or particle label.
• the detection technique can be performed with the biosensor surface either immersed in fluid or dried.
• a colorimetric resonant reflectance biosensor When a colorimetric resonant reflectance biosensor is illuminated with collimated white light and reflects only a narrow band of wavelengths, or a single band of wavelengths is reflected.
• the narrow wavelength band is described as a wavelength "peak.”
• the "peak wavelength value” (PWV) changes when molecules are deposited or removed from the biosensor surface.
• a readout instrument illuminates distinct locations on the biosensor surface with collimated white light, and collects collimated reflected light. The collected light is gathered into a wavelength spectrometer for determination of PWV.
• Evanescent wave-based biosensors can comprise a waveguiding film supported by a substrate; between the waveguiding film (and optionally as part of the substrate) is a diffraction grating. See, e.g., U.S. Pat. No. 4,815,843.
• a low-k dielectric material such as low-k nanoporous material can be used for the diffraction grating or the combined low- k nanoporous material and substrate.
• the waveguide comprises waveguiding film and the substrate.
• the waveguiding film can be, e.g., tin oxide, tantalum pentoxide, zinc sulfide, titanium dioxide, silicon nitride, or a combination thereof, or a polymer such as polystryrole or polycarbonate.
• a diffraction grating exists at the interface of the waveguiding film and the substrate or in the volume of the waveguiding film.
• the diffraction grating comprises a low-k material, such as low-k nanoporous material.
• the refractive index of the waveguiding film is higher than the index of the adjacent media (i.e., the substrate and the test sample).
• the substrate can be, e.g., plastic, glass or epoxy.
• a specific binding substance can be immobilized on the surface of the waveguiding film and a test sample added to the surface.
• Laser light propagates in the waveguiding film by total internal reflection. Changes in refractive index of the waveguiding film caused by molecules binding to it can be detected by observing changes in the angle of the emitted, out-coupled light.
• a biosensor of the invention can comprise an inner surface, for example, a bottom surface of a liquid-containing vessel.
• a liquid-containing vessel can be, for example, a microtiter plate well, a test tube, a petri dish, or a microfluidic channel.
• One embodiment of this invention is a biosensor that is incorporated into any type of microtiter plate.
• a biosensor can be incorporated into the bottom surface of a microtiter plate by assembling the walls of the reaction vessels over the resonant reflection surface, so that each reaction "spot" can be exposed to a distinct test sample. Therefore, each individual microtiter plate well can act as a separate reaction vessel. Separate chemical reactions can, therefore, occur within adjacent wells without intermixing reaction fluids, and chemically distinct test solutions can be applied to individual wells.
• the most common assay formats for pharmaceutical high-throughput screening laboratories, molecular biology research laboratories, and diagnostic assay laboratories are microtiter plates.
• the plates are standard-sized plastic cartridges that can contain 96, 384, or 1536 individual reaction vessels arranged in a grid. Due to the standard mechanical configuration of these plates, liquid dispensing, robotic plate handling, and detection systems are designed to work with this common format.
• a biosensor of the invention can be incorporated into the bottom surface of a standard microtiter plate.
• biosensor surface can be fabricated in large areas, and because the readout system does not make physical contact with the biosensor surface, an arbitrary number of individual biosensor areas can be defined that are only limited by the focus resolution of the illumination optics and the x-y stage that scans the illumination/detection probe across the biosensor surface.
• Phage are bacterial viruses and are species specific. For this particular discussion, two Escherichia coli phage, Ml 3 and Lambda, are being discussed. In the case of other bacterial phage and/or mammalian viruses the same premise applies.
• a population of phage in particular, a non-homogenous, crude, and/or unconcentrated population of phage, such as a phage display library can be used in methods of the invention.
• a non- homogenous preparation of phage comprises a preparation that contains one or more type of phage, e.g., a phage display library wherein each phage displays a different binding molecule.
• a crude phage preparation is a preparation that contains one or more types of phage in the medium in which bacteria infected with phage was grown in. In the case of Ml 3, the phage fuse through the membrane and the cell is not lysed. In the case of lambda, the cells are lysed and the medium would contain cellular components. In this case the crude phage preparation would be clarified of bacterial cells and membrane components by centrifugation.
• a crude phage preparation contains one or more types of phage at low concentrations in the presence of media components and excreted cellular catabolites.
• An unconcentrated phage preparation is a phage preparation where the phage has not been precipitated.
• the medium is removed and the phage can be resuspended in a defined buffer such as PBS.
• the phage is usually precipitated with a PEG solution one or two times and is stored in PBS glycerol.
• the phage is resuspended in smaller volumes of buffer than the original volume of medium.
• a typical 1-2 liter culture of medium will be resuspended in a final volume of about 2-5 ml of PBS, providing a purified and concentrated stock of phage.
• a phage preparation is immobilized to a biosensor.
• a phage preparation can be passively immobilized to a biosensor surface.
• the phage surface can be blocked and an antigen (e.g. small molecule, carbohydrate, polymer, peptide, soluble protein, antigen mimic of a cellular receptor, or mammalian cells) can be screened for binding to the immobilized phage.
• the binding of the antigen specifically to the phage is measured by the change in signal generated by such a binding event, typically via optical, electrical or visual means.
• Antigen binding to the phage can be ranked by concentration of the phage, off-rate of the antigen, and the ability of the phage to functionally bind cells.
• a phage preparation can be immobilized to a biosensor surface using specific antibody immobilization.
• An antibody to a phage coat protein is immobilized to the biosensor surface.
• the antibody can be passively immobilized to the biosensor surface or via a specific surface such as protein A or a protein A plus anti-Fc surface.
• the surface can be blocked by a blocker.
• the binding of the phage specifically to the surface is measured by the change in signal generated by such a binding event, typically via optical, electrical or visual means.
• the display on the phage (virus) can be a peptide, small protein, and/or an antibody fragment or non-existent.
• the binding of the cognate ligand is then sequentially measured by the change in signal generated by such a binding event, typically via optical, electrical or visual means.
• the ligand can be, e.g., a small molecule, carbohydrate, polymer, peptide, soluble protein, antigen mimic of a cellular receptor or a protein on the surface of cells.
• the protein expressed on the surface of the cell can be, e.g., a membrane-associated protein, a single or multi-transmembrane protein, or a protein channel.
• a phage preparation can be immobilized to a biosensor surface by an antigen bound to the biosensor surface.
• An antigen such as a small molecule, carbohydrate, polymer, peptide, soluble protein, or antigen mimic of a cellular receptor
• the antigen surface can be blocked.
• the binding of the phage preparation specifically to the antigen is measured by the change in signal generated by such a binding event, typically via optical, electrical or visual means.
• the display on the phage can be, e.g., a peptide, small protein, and/or an antibody fragment. Phage binding to the antigen can be ranked by concentration of the antigen, and the off-rate of the phage.
• Antibody fragments (and proteins) can be captured specifically to the surface of the biosensor through biotin or proteinaceous tags (multiple histidines, c-myc, or FLAG, MBP, GST_etc.) fused to their N-or C-terminal domains.
• a specific surface can be built on the biosensor based on antibodies to the tags.
• antibody fragments can be captured specifically through the constant region (CHl).
• a specific surface can be built on the biosensor based on antibodies to this region using anti-lambda, anti-kappa, a mixture of anti-lambda and anti-kappa, and/or anti-F(ab') 2 antibodies. The surface can be blocked.
• the binding of the antibody fragment specifically to the antibody is measured by the change in signal generated by such a binding event, typically via optical, electrical or visual means.
• the immobilization of the antibody fragment can be from a pure or crude (whole cell extract, periplasmic extract, or spent media) sample.
• the binding of the cognate ligand can then be sequentially measured by the change in signal generated by such a binding event, typically via optical, electrical or visual means.
• the ligand can be, e.g., a small molecule, carbohydrate, polymer, peptide, soluble protein, antigen mimic of a cellular receptor or a protein on the surface of cells.
• the protein expressed on the surface of the cell can be, e.g., a membrane-associated protein, a single or multi- transmembrane protein, or a protein channel.
• detectable labels can be used to detect specific binding substances or binding partners on the surface of a biosensor.
• detectable labels can still comprise other types of labels and markers for enhancement of assay sensitivity, immobilization of specific binding partners to a biosensor surface, enhancement of binding or hybridization of specific binding substances to their binding partners, and for other purposes.
• Molecules can be immobilized onto a biosensor so that they will not be washed away by rinsing procedures, and so that binding to molecules in a test sample is unimpeded by the biosensor surface.
• surface chemistry strategies have been implemented for covalent attachment of molecules to, for example, glass for use in various types of microarrays and biosensors. These same methods can be readily adapted to a biosensor of the invention.
• One or more types of molecules can be attached to a biosensor surface by physical adsorption ⁇ i.e., without the use of chemical linkers) or by chemical binding ⁇ i.e., with the use of chemical linkers). Chemical binding can generate stronger attachment of molecules on a biosensor surface and provide defined orientation and conformation of the surface-bound molecules.
• amine activation examples include, for example, amine activation, aldehyde activation, and nickel activation. These surfaces can be used to attach several different types of chemical linkers to a biosensor surface. While an amine surface can be used to attach several types of linker molecules, an aldehyde surface can be used to bind proteins directly, without an additional linker. A nickel surface can be used to bind molecules that have an incorporated histidine (“his”) tag. Detection of "his-tagged" molecules with a nickel-activated surface is well known in the art (Whitesides, Anal. Chem. 68, 490, (1996)).
• his histidine
• Immobilization of specific binding substances to plastic, epoxy, or high refractive index material can be performed essentially as described for immobilization to glass. However, an acid wash step can be eliminated where such a treatment would damage the material to which the specific binding substances are immobilized.
• a specific binding substance can be immobilized on a biosensor by for example, physical adsorption or by chemical binding.
• a specific binding substance can be, for example, an organic molecule, such as a nucleic acid, polypeptide, antigen, polyclonal antibody, monoclonal antibody, single chain antibody (scFv), F(ab) fragment, F(ab') 2 fragment, Fv fragment, small organic molecule, cell, virus, phage, bacteria, polymer, peptide solutions, single- or double-stranded DNA solutions, RNA solutions, solutions containing compounds from a combinatorial chemical library, or biological sample; or an inorganic molecule.
• an organic molecule such as a nucleic acid, polypeptide, antigen, polyclonal antibody, monoclonal antibody, single chain antibody (scFv), F(ab) fragment, F(ab') 2 fragment, Fv fragment, small organic molecule, cell, virus, phage, bacteria, polymer, peptide solutions, single- or
• a biological sample can be for example, blood, plasma, serum, gastrointestinal secretions, homogenates of tissues or tumors, synovial fluid, feces, saliva, sputum, cyst fluid, amniotic fluid, cerebrospinal fluid, peritoneal fluid, lung lavage fluid, semen, lymphatic fluid, tears, or prostatitc fluid.
• an antibody, an antibody fragment, an antigen, or a phage is immobilized on a biosensor.
• Such specific binding substances can be directly immobilized as described herein, or can be indirectly immobilized via a specific surface.
• an antibody or protein comprising a proteinaceous tag e.g.
• a binding partner is a substance that specifically binds to a specific binding substance.
• a binding partner can be any type of sample or molecule as described above for specific binding substances.
• the methods of the invention can be used, for example, in: development of therapeutic and diagnostic antibodies by screening hybridomas, human antibodies from mice and phage display technologies; screening mRNA T7 phage display libraries; and a mammalian and bacterial cell free system for titration of bacterial as well as mammalian viruses.
• binding molecules will be discussed, in particular, antibody fragments and/or full antibodies, eg. Figure 1.
• the method of the invention can be used to titrate viruses.
• titration of viruses requires live mammalian and bacterial cells for mammalian and bacterial viruses, respectively.
• the circles of death or slower growing cells are counted.
• antibodies to the coat protein of the mammalian or bacterial virus are attached to the biosensor and dilutions of intact virus are detected via the antibody and coat protein interaction.
• Ml 3 phage can be titrated by direct immobilization of the phage using a GA3 high density gluteraldehyde biosensor, see eg. Figure 2.
• the GA3 biosensor was hydrated with PBS for 30 minutes, then purified Ml 3 in 20% glycerol were prepared using 1:2 serial dilutions from 1.0el4 to 1.3el l plaques per ml and were added to the sensor in 20% glycerol and PBS.
• the phage solutions were incubated with the sensor for 2 hours. The unbound phage were washed away and an endpoint reading was recorded for the amount of phage immobilized via the free amines on the phage protein coat.
• Methods of the invention can also be used in the investigation of the biology (receptor binding, entry into cells, screening of neutralizing antibodies, etc.) surrounding mammalian viruses.
• a virus is immobilized on the biosensor and antibodies or proteins are screened for their ability to increase or decrease the fusion of the virus to the mammalian cells.
• the methods of the invention can also be used to detect toxins, viruses, and other bio-terrorism agents.
• targets such as known toxins, virus, and other bio-terrorism agents or antigens derived from them with a phage display library. These libraries can display peptides, antibody fragments, protein scaffolds, or protein domains. During the panning process, phage are enriched for the ability to bind the target.
• methods of the invention can be used to detect the toxins, virus, or other bio- terrorism agents by immobilizing the phage to the biosensor. This would replace the cumbersome work of synthesizing the peptide or performing molecular cloning of the protein or antibody fragments that are displayed on the phage.
• Methods of the invention can be used for screening (after a phage panning or selection experiment) a large number of potential binding display phage for positive binding. Due to the ability to create specific binding surfaces, phage can be specifically pulled out of defined and undefined solutions, such as bacterial extracts and spent media and immobilized on a biosensor.
• Methods of the invention can also be used to rank antibody affinities early at the phage level of screening, when using phage display technologies to identify therapeutic or diagnostic antibodies.
• a phage displaying an antibody fragment can be immobilized to the biosensor directly or via an antibody to the coat protein.
• a signal for the amount of phage immobilized per well is recorded.
• the antigen for the antibody displayed on the phage is added and a signal is recorded.
• a rank can be determined by dividing the signal/well for antigen by the signal/well for the phage. By this calculation the antigen signal is normalized for units of phage in each assay (well).
• the antibody fragment on the phage could be ranked for its monovalent affinity without the use of molecular biology techniques to clone the gene for the antibody fragment. This technique would enable ranking of antibody fragments with out the need for cloning the gene, expression of the antibody fragment, purification of the antibody fragment, then ranking of binding with the antigen.
• Methods of the invention can be used to determine the off-rate of the display on a phage for the corresponding antigen. For example, a phage is immobilized on a biosensor, washed, and antigen is bound. After each wash sequence, the biosensor is read. If the antigen is released from the phage on the biosensor, then there is a loss in signal. The biosensor is monitored over multiple washes and the off-rate is calculated as the loss of signal over time.
• Binding partners can be identified using this technology, by binding cellular components to the biosensor, then adding protein partners or phage containing DNA sequences corresponding to cellular components.
• a test sample such as cell lysates containing binding partners, can be applied to a biosensor of the invention, followed by washing to remove unbound material.
• the binding partners that bind to a biosensor can subsequently be eluted from the biosensor and identified by, for example, mass spectrometry.
• a phage DNA display library can be applied to a biosensor of the invention followed by washing to remove unbound material. Individual phage particles bound to the biosensor can be isolated and the inserts in these phage particles can then be sequenced to determine the identities of the binding partners.
• Antibodies can be immobilized in an array format onto a biosensor, which is then contacted with a test sample of interest comprising binding partners, such as proteins. Only the proteins that specifically bind to the antibodies immobilized on the biosensor remain bound to the biosensor.
• binding partners such as proteins.
• Such an approach is essentially a large-scale version of an enzyme-linked immunosorbent assay; however, the use of an enzyme or fluorescent label is not required.
• the methods of the invention provide several advantages over conventional phage display and phage panning protocols, including, for example: (1) label free and direct binding of the display on the phage immobilized on the biosensor to cells or other binding partners is not influenced by protein labels and amplification of secondary signals; (2) specific pull down of phage (i.e., immobilization of phage to a biosensor) from crude samples and the binding of cells or other binding partners without excessive washing detects functional binding of mammalian cells faster; and (3) high throughput, thereby providing the most efficient and rapid phage screening methods.
• the methods of the invention enable a researcher to acquire critical information early, thereby accelerating therapeutic discoveries. In many cases the methods of the invention could save discovery researchers up to three to six months of effort in identifying therapeutic antibodies.
• Biosensors of the invention are also capable of detecting and quantifying the amount of a binding partner from a sample that is bound to a biosensor array distinct location by measuring the shift in reflected wavelength of light. Additionally, the wavelength shift at one distinct biosensor location can be compared to positive and negative controls at other distinct biosensor locations to determine the amount of a binding partner that is bound to a biosensor array distinct location.
• Methods of the invention can be used to capture antibodies and antibody fragments, from complex media such as, e.g., periplasmic extracts, hybridoma supernatants, plasma, or sera. See, e.g., Figures 3 to 10.
• the following specific surface can be modified to capture full IgGs or F(ab) by substituting rabbit anti-mouse-Fc, rabbit anti-human Fc, rabbit anti-human F(ab')2, or the rabbit anti-mouse-F(ab')2 in the following example.
• the following procedure is for the capture of sF(ab) spiked into PBS and periplasmic cultures described in Figure 3.
• a specific capture surface for sFab was created on a SRU BIND® TIO Biosensor, Figure 3(A).
• a hydrated TIO BIND® Biosensor was coated with 20 ug/ml Protein A in PBS for 30 minutes, washed and an endpoint reading was recorded for the amount of protein A deposited. The Biosensor was then blocked for 30 minutes with 1% milk, washed and an endpoint reading was recorded for the amount of milk deposited. Then 20 ug/ml of the specific capture reagent, rabbit anti-mouse F(ab')2 specific IgG, was incubated for 30 minutes, washed, and an endpoint reading was recorded for the amount of rabbit anti-mouse F(ab')2 specific IgG deposited. The remaining protein A sites were blocked with 50 ug/ml rabbit IgG for 30 minutes.
• mouse sF(ab) a specific capture surface for mouse sF(ab) has been created.
• purified polyclonal mouse sF(ab), 0.33, 1.0 and 3.0 ug/ml were spiked into PBS and into a Escherichia coli periplasmic prep, not expressing sF(ab), Figure 3(A) and 4(B).
• FIG. 4 shows scFv capture from periplasmic extract on a TIO BIND® Biosensor.
• a hydrated TIO BIND® Biosensor was coated with 20 ug/ml Protein A in PBS for 30 minutes, washed and an endpoint reading was recorded for the amount of protein A deposited. The Biosensor was then blocked for 30 minutes with 1% milk, washed and an endpoint reading was recorded for the amount of milk deposited. Then 20 ug/ml of the specific capture reagent, rabbit anti-mouse-Fc, was incubated for 30 minutes, washed, and an endpoint reading was recorded for the amount of rabbit anti-mouse-Fc specific IgG deposited.
• the specific capture reagent rabbit anti-mouse-Fc
• Figure 5 shows scFv capture from periplasmic extract on a SAl BIND® Biosensor.
• Eight micrograms per milliliter of scFv was spiked into PBS and periplasmic extracts of Escherichia coli, not containing plasmids encoding scFv. The scFv were incubated for 1 hour, washed, and an endpoint reading was recorded for the amount of scFv deposited.
• the amount of scFv is tabulated in Figure 5(B) and graphically represented in Figure 5(C).
• Figure 6 shows scFv capture from periplasmic extract on a GAl BIND®
• Biosensor A hydrated GAl BIND® Biosensor, gluteraldehyde, was incubated with 50 ug/ml anti-cmyc for one hour, washed, and an endpoint reading was recorded for the amount of anti-myc deposited, Figure 6(A). Two hundred micrograms per milliliter of neutravidin was incubated with the BIND® Biosensor to block any remaining gluteraldehyde reactive groups for 1 hour, washed, and an endpoint reading was recorded for the amount of neutravidin deposited. Five micrograms per milliliter of scFv was spiked into PBS and periplasmic extracts of Escherichia coli, not containing plasmids encoding scFv.
• mouse IgGs created via hybridoma technologies are captured from hybridoma supernatants then tested for their ability to bind soluble antigen and antigen expressed on cells.
• a specific capture surface for mouse IgG was created on a SRU BIND® TIO Biosensor, Figure 7(A) and 7(B).
• a hydrated TIO BIND® Biosensor was coated with 20 ug/ml Protein A in PBS for 30 minutes, washed and an endpoint reading was recorded for the amount of protein A deposited. The biosensor was then blocked for 30 minutes with 1% milk, washed and an endpoint reading was recorded for the amount of milk deposited.
• Figure 7(D) shows the antigen and cell binding to the captured IgGs on the BIND® Biosensors.
• Figure 7(E) shows the nm shifts for the antigen and cell binding as well as the ratio of antigen signal divided by the IgG signal and the ratio of the cell binding signal divided by the IgG signal. These ratios can be used to rank the IgGs.
• Antibodies 22, 25, 26, 28, 30, 31, 34, 36 and 37 can be classified as antigen binders (antigen binding divided by IgG binding) with 37 having the highest affinity for the antigen with a ratio of 0.13.
• Antibodies 22, 25, and 26 have similar affinities and constitute the second class of binders with a ratio of 0.10.
• the remaining antibodies (28, 30, 31, 34 and 36) have much lower affinities with their ratio less than 0.04.
• the IgGs can also be classified as a general cell binder in the case of antibody 30 with a ratio for the parental cell line divided by IgG of 0.11 and the antigen presenting cells divided by IgG of 0.16.
• the second cell binding class would be specific for antigen expressed on the cells as in the antibodies 25 and 26 with ratios for the antigen presenting cells divided by IgG of 0.49 and 0.30, respectively, and the parental cell line divided by IgG of 0.00 and 0.01, respectively.
• the rest of the antibodies are non-cell binders with ratios for binding the parental cell line divided by IgG and the antigen presenting cells divided by IgG less than 0.04.
• FIG 8 shows antibody capture from serum using an anti-Fc TIO BIND® Biosensor.
• a specific capture surface for mouse IgG was created on a SRU BIND® TIO Biosensor Figure 8(A).
• a hydrated TIO BIND® Biosensor was coated with 20 ug/ml Protein A in PBS for 30 minutes, washed and an endpoint reading was recorded for the amount of protein A deposited.
• the Biosensor was then blocked for 30 minutes with 1% milk, washed and an endpoint reading was recorded for the amount of milk deposited.
• 20 ug/ml of the specific capture reagent, rabbit anti-mouse Fc was incubated for 30 minutes, washed and an endpoint reading was recorded for the amount of rabbit anti- mouse Fc deposited.
• Figures 8(B) and (C) shows the capture of mouse IgG (serial dilution from 0.1 - 6.4 ug/ml) that were spiked into PBS and 5%, 10%, 20%, 40% and 100% serum.
• Figures 8(D) and 8(E) are the graphical representation of the tabulated data in Figures 8(B) and 8(C).
• Figure 9 shows the Drug - anti-Drug assay in serum using a GAl Bind Biosensor.
• An IgG is the mimic for a therapeutic antibody drug and the anti-Drug is a F(ab')2 spiked into PBS and mouse sera to mimic IgGs found in human sera from clinical isolates using a GAl BIND® Biosensor.
• a hydrated GAl BIND® Biosensor was incubated with 20 ug/ml IgG for 1 hour, washed and an endpoint reading was recorded for the amount IgG deposited on the surface.
• the free aldehydes remaining on the surface of the GAl BIND® Biosensor were bound up by incubating the sensor with 100 ug/ml non-immune rabbit IgG for 2 hours.
• the IgG (Drug) rlgG surface was also blocked by START Block from Pierce for 1 hr.
• Figure 9(A) shows the nm shifts for the production of a drug surface specific.
• a titration curve of anti-Drug from 0 - 7.1 ug/ml was incubated on the Biosensor by diluting the anti-drug in serial 1:3 dilutions into PBS or 11%, and 30% mouse serum.
• the binding of anti-Drug to the 20 ug/ml, Figure 9(B) and to 0 ug/ml Drug surface, Figure 9(C) is represented in graphs.
• the same amount of anti-drug is captured in 11% serum compared to PBS. Less anti-Drug is captured in 30% serum, but the anti-Drug is detectable.
• Figure 10 shows the development of a Drug - anti-Drug assay in serum using a TIO BIND® Biosensto.
• An IgG is the mimic for a therapeutic antibody drug and the anti- Drug is a F(ab')2 spiked into PBS and mouse sera to mimic IgGs found in human sera from clinical isolates.
• Figure 10(A) shows the shifts measured during the creation of a specific surface for the capture of mouse IgGs.
• a hydrated TIO BIND® Biosensor is coated with 20 ug/ml of protein A.
• One percent milk is used to block any remaining binding sites on the TIO not filled by protein A. After the milk blocking step the surface is incubated with 20 ug/ml rabbit anti-human-Fc.
• Rabbit IgG at 50 ug/ml is used to block any protein A binding sites not filled by the rabbit anti-mouse-Fc.
• each reagent is incubated with the surface for thirty minutes, the surface is washed and an endpoint reading was recorded for the amount of each reagent deposited on the surface.
• Fifty micrograms per milliliter of Drug (IgG) was incubated with the specific surface for 1 hour, washed and an endpoint reading was recorded for the amount of each Drug deposited on the surface.
• a titration curve of anti-Drug from 0 — 7.1 ug/ml was incubated on the Biosensor by diluting the anti-drug in serial 1:3 dilutions into PBS or 11%, and 30% mouse serum.
• the binding of anti-Drug to the 50 ug/ml and 0 ug/ml Drug surface is shown in Figure 10(B) and H(C), respectively. Less anti-Drug is captured in 11% and 30% serum, but the anti-Drug is detectable. Determination of epitope classes within an antibody screen
• Epitope classes within an antibody screen (antibody or antibody fragment) or a phage screen can be determined using methods of the invention.
• the binding molecules to be classified can be antibodies or display phage or a combination of both.
• the first binding molecule is passively or specifically immobilized to a biosensor.
• the biosensor comprises the bottom surface of a microtiter plate. The surface can be blocked by blockers.
• An antigen (such as a carbohydrate, polymer, peptide, soluble protein, or antigen mimic of a cellular receptor) is captured specifically by the display phage, antibody, or antibody fragment surface. Individual phage clones, antibodies, or antibody fragments to be classified are added to the wells.
• FIG. 11 shows an endpoint analysis of monoclonal antibody binding target peptides for epitope mapping using a colorimetric resonant reflectance biosensor.
• Figure H(A) shows the shifts measured during the creation of a specific surface for the capture of mouse IgGs.
• a hydrated TIO BIND® Biosensor is coated with 20 ug/ml of protein A.
• the protein A acts as a capture surface for the rabbit anti-mouse-Fc that will specifically captures mouse IgGs.
• One percent milk is used to block any remaining binding sites on the TIO not filled by protein A.
• the surface is incubated with 20 ug/ml rabbit anti-mouse-Fc.
• Rabbit IgG at 50 ug/ml is used to block any protein A binding sites not filled by the rabbit anti-mouse-Fc.
• each reagent is incubated with the surface for thirty minutes, the surface is washed and an endpoint reading was recorded for the amount of each reagent deposited on the surface.
• Figure 11 (B) the capture of the four mouse IgGs by the protein A : milk : rabbit anti- mouse-Fc : rabbit IgG surface is recorded. Ten micrograms per milliliter of IgGs were incubated with the specific surface for 30 minutes. To ensure that the second layer of mouse IgGs bind through the antigen and not the rabbit anti-mouse-Fc, non-immune mouse IgG from Pierce is added at 50 ug/ml to fill any unoccupied rabbit anti-mouse-Fc binding sites, Figure H(A).
• Figure 11 (C) records the shift measured when antigen is added to captured mouse IgG.
• Ab-4 does not bind antigen and the 4 IgGs can be classified as antigen binders (Ab-I, Ab-2 and Ab-3) and antigen non-binders (Ab-4).
• the antigen binding antibodies can also be ranked by dividing the antigen shift by the antibody shift measured by the BIND® Biosensor.
• Ab-2 has the highest affinity for antigen with a ratio of 0.56.
• Ab-3 has a slightly higher affinity for the antigen than Ab-I with ratios of antigen binding per antibody shift of 0.38 and 0.35, respectively.
• Figure H(D) shows the grid created by the addition of the same mouse IgGs in layer 1 as layer 3. If two IgGs in the same well bind the same area of the antigen, then no signal will be measured.
• Ab-2 is in a second binding class and can be used as a sandwich partner for both Ab-I and Ab-3: Ab-I vs. Ab-2 equals 0.138; Ab-2 vs. Ab-I equals 0.150; Ab-3 vs. Ab-2 equals 0.203; and Ab-2 vs. Ab-3 equals 0.212.
• This technique will work where the first and/or the second antibody immobilized are a displayed on phage or a soluble antibody (purified or crude sample). Combinations include phage : phage, phage : antibody fragment, antibody fragment : phage, antibody fragment : antibody fragment, full antibody : phage, phage : full antibody, full antibody : full antibody, antibody fragment : full antibody, and antibody : antibody fragment.
• This can also be applied to hybridoma and phage display screens as well as human antibodies made in other hosts, such as mice.

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