US 2011 0230644A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0230644 A1 Jablonski et al. (43) Pub. Date: Sep. 22, 2011

(54) MICROBUBBLES FORAFFINITY Publication Classification SEPARATION (51) Int. Cl. (76) Inventors: Edward Jablonski, Escondido, CA C07K L/00 (2006.01) (US); Thomas Adams, Rancho Santa Fe, CA (US) (52) U.S. Cl...... S30/362

(22) Filed: May 31, 2011 The present invention relates to methods, compositions and kits for affinity isolation, affinity purification and affinity Related U.S. Application Data assay based on microbubbles coated with an affinity mol (62) Division of application No. 1 1/718,090, filed on Mar. ecule. Particularly, the invention provides protein 13, 2009, filed as application No. PCT/US05/40162 on microbubbles coated with an affinity molecule. In addition, Nov 3 2005 pp the invention provides glass microbubbles coated with an • - s affinity molecule. Methods of using the microbubbles of the (60) Provisional application No. 60/624,948, filed on Nov. invention for isolating analytes and cells are specifically pro 3, 2004. vided. US 2011/023.0644 A1 Sep. 22, 2011

MICROBUBBLES FORAFFINITY microbubbles, such as albumin microbubbles. The protein SEPARATION microbubbles may beformed by the introduction of a gas into a solution of protein, for example, by Sonication. In one FIELD OF THE INVENTION aspect of the invention, gas may be introduced into the protein 0001. This invention relates to methods, compositions and through a process comprising heating a solution of protein. In kits for affinity isolation, affinity purification, and affinity another embodiment, the protein microbubbles may be stabi assay based on microbubbles coated with an affinity mol lized, for example, by denaturing the protein or treatment ecule. with Cr. 0008. In another embodiment of the invention, the BACKGROUND OF INVENTION microbubbles are glass microbubbles. In one aspect of the invention, the glass microbubbles have a density of about 0.6 0002 The field of isolating cells, virus, bacteria and g/cc and an average diameter of about 30 Jum. soluble molecules has used various types of particles as a 0009. According to the invention, the affinity molecule solid phase to absorb or bind to the target of interest. For can be a receptor, a ligand, or an antibody. Alternatively, the example, magnetic particles, when coated with a ligand, Such affinity molecule can be biotin, avidin or streptavidin. as an antibody, can bind to a target cellor soluble protein. The 0010. The affinity molecule may be directly coupled to the bound target on the magnetic particle effects a separation of microbubble, for example, by using a heterobifunctional the target from other cells types or proteins. There have been reagent such as SulfoSuccinimidyl 4-(N-maleimidomethyl) various improvements on this original work and commercial cyclohexane-1-carboxylate. In another embodiment of the examples have been available for years. invention, the affinity molecule is indirectly coupled to the 0003. Others have used latex particles, liposomes, milkfat microbubble, such as through the interaction of at least one globules, plastic particles such as polystyrene and polyethyl other molecule. In one aspect of the invention, the ene and polypropylene, nylon etc. As capture Supports, each microbubble is directly coupled to streptavidin and the affin of these has its own particular attributes and problems. Non ity molecule is biotinylated, such that the streptavidin and specific binding of non-target cells and proteins is the most biotin interact to couple the affinity molecule to the common problem encountered in these methods, resulting in microbubble. imperfect separation. Additionally, most methods require at 0011. According to one embodiment of the invention, the least one additional step following binding to effect separa affinity molecule may be directly coupled to the microbubble tion of unbound species from the particle-bound, e.g. for through an epoxy coating on the microbubble. In another magnetic particles a magnetic field must be applied, centrifu embodiment, the affinity molecule is coupled through an gation is sometimes used to separate the particles from Solu amine functional group on the microbubble. tion or filtration of the particles from solution. 0012. In one aspect of the invention, glass microbubbles 0004. In general, the time required to bind the target cells are treated to generate reactive surface residues, which are in or proteins is related to the surface area of the particles and the turn reacted with 3-aminopropyltriethoxy silane, generate quantity of particles per unit Volume of Solution. The Smaller amines. In another aspect, glass microbubbles are cis-diol the particle the more rapid the binding due to increased Sur coated and the affinity molecule is directly coupled to the face area Unfortunately, greater Surface area generally glass through the cis-diol coating. The cis-diol coating can be increases non-specific binding. generated, for example, by treating the glass microbubbles to 0005 Separation methods may, depending on the nature generate reactive Surface hydroxyl residues, reacting the or principle of the separation, co-isolate different particles. hydroxyl residues with 3-glycidoxypropyltrimethoxysilane This is apparent in separations based on gravity centrifuga to generate epoxy functional residues, and treating the epoxy tion in which particles and cells or other species may co functional residues with acid to convert the epoxy function to pellet. In addition some cell types such as macrophages and cis-diol functions. monocytes may themselves non-specifically ingest the par 0013 The present invention also provides a method for ticles and can be isolated along with the target cells. While generating protein microbubbles for use in affinity isolation individual limitations of the previous technology may be or affinity assay. In one aspect, the method comprises heating minimized or avoided by taking certain steps or precautions, a solution of protein. In another aspect of the method, a certain limitations are inherent and cannot be entirely over Solution of protein is treated ultrasonically to introduce gas come. The fact that there are many different approaches with into the Solution, thereby generating protein microbubbles. In varying degrees of Success suggests that a better Solution to yet another aspect of the method, the solution of protein is the problem is needed. treated mechanically in the presence of a gas or a gas mixture. 0006. The optimal separation agent would have an infinite 0014. In alternative aspects of the invention, the gas is air Surface area with Zero non-specific interaction so that the and the protein is albumin. The protein microbubbles may be binding would occur instantaneously and minimize binding stabilized, for example by treatment with Cr". to non-target cells or soluble molecules. Ideally, the agent 0015 The present invention also provides a method for should separate itself from the cell suspension or soluble generating microbubbles for use in affinity isolation or affin moleculesolution without entrapping non-target cells or mol ity assay comprising providing microbubbles; and coating the ecules respectively. microbubbles with an affinity molecule. 0016. In another embodiment of the invention, methods SUMMARY OF THE INVENTION for affinity isolation or affinity assay of a species are provided. 0007. The present invention provides compositions for use According to these embodiments, the method comprises the in affinity isolation or affinity assay comprising microbubbles steps of providing microbubbles coated with an affinity mol that are covalently coated with an affinity molecule. In one ecule in a solution, contacting the microbubbles with a spe embodiment of the invention, the microbubbles are protein cies that interacts with the affinity molecule in a solution, US 2011/023.0644 A1 Sep. 22, 2011

thereby generating microbubbles coated with the species, and 0024 “Bubble,” as used herein refers to a small, hollow allowing the microbubbles coated with the species to float to and lightweightglobule, typically a small spherical Volume of the top of the solution, thereby separating the species from the gas encased within a thin film. Bubbles can be filled with any Solution. gas, including, but not limited to oxygen, nitrogen, carbon 0017. In one aspect of this embodiment, the species is a dioxide, helium, fluorocarbon gases and various combina receptor, a ligand, or an antigen. In one embodiment, the tions thereof, such as air. The thin film may be any material species is an analyte. In alternative embodiments, the species that can encase a small Volume of gas, Such as an insoluble is a virus or a cell. protein or lipid; a polymeric or non polymeric material; a 0018. In another aspect of the invention, protein Solid such as a metal; a Solid glass, ceramic or similar mate microbubbles, particularly albumin microbubbles can be rial; or a plastic, such as polystyrene, polyethylene, polypro treated with detergent, pressure or vacuum to release the pylene, nylon, etc. In a preferred embodiment, the thin film is species. albumin. In another preferred embodiment, the thin film is borosilicate glass. In one embodiment, the thin film is stable under the conditions and Solutions it is exposed to. In another DETAILED DESCRIPTION OF THE INVENTION embodiment, the bubble can be selectively burst, crushed or 0019. It is to be understood that both the foregoing general solubilized. description and the following detailed description are exem (0025 “Microbubbles” are small bubbles, generally in the plary and explanatory only and are not restrictive of the inven range of 0.1 to 100 microns, typically 1 to 50, and frequently tion claimed. As used herein, the use of the singular includes 2 to 20 or 2 to 30 microns in diameter. the plural unless specifically stated otherwise. As used herein, 0026. The term “analyte, as used herein, refers to any “or means “and/or unless stated otherwise. Furthermore, Substance that it is desirable to detect in an assay, and which use of the term “including” as well as other forms, such as may be present in a sample. The analyte can be, without “includes, and “included,” is not limiting. As used herein, limitation, any substance. In a preferred embodiment of the “can means “may unless stated otherwise. invention, an analyte comprises a Substance for which there 0020. The section headings used herein are for organiza exists a naturally occurring antibody or for which an antibody tional purposes only and are not to be construed as limiting can be prepared. The analyte may, for example, be a protein, the subject matter described. All documents, or portions of a polypeptide, a hapten, a carbohydrate, a lipid, a drug, a cell, documents, cited in the application including, but not limited a cellular Subcomponent or organelle (e.g., lysozomes, mito to, patents, patent applications, articles, books, manuals, and chondria) or any other of a wide variety of biological or treatises are hereby expressly incorporated by reference in non-biological molecules, complexes or combinations their entirety for any purpose. thereof. In another embodiment, the analyte is an antibody. In still another embodiment, the analyte is a nucleic acid (DNA, Definitions RNA, PNA and nucleic acids that are mixtures thereof or that include nucleotide derivatives or analogs) 0021. Before proceeding further with a description of the 0027 Polyvalent ligand analytes that can be detected specific embodiments of the present invention, a number of using compositions, methods and kits of the present invention terms will be defined and described in detail. will normally be poly(amino acids), i.e., polypeptides and 0022. Unless specific definitions are provided, the nomen proteins, polysaccharides, nucleic acids, and combinations clatures utilized in connection with, and the laboratory pro thereof. Such combinations include components of cells, tis cedures, techniques and methods described herein are those Sues, bacteria, viruses, cell walls, cell membranes, cellular known in the art to which they pertain. Standard chemical organelles, chromosomes, genes, mitochondria, nuclei and symbols and abbreviations are used interchangeably with the the like. According to one aspect of the invention, certain full names represented by Such symbols. Thus, for example, analytes do not contain nucleic acid. the terms “carbon' and “C” are understood to have identical 0028. A wide variety of protein analytes may be advanta meaning. Standard techniques may be used for chemical Syn geously detected using the methods of the present invention. theses, chemical modifications, chemical analyses, pharma Such protein analytes can be classified according to family, ceutical preparation, formulation, delivery, and treatment of with each family having similar structural features, biological patients. functions, relationship to specific microorganisms (particu 0023 Standard techniques may be used for recombinant larly disease causing microorganisms), and the like. Protein DNA methodology, oligonucleotide synthesis, tissue culture families of particular interest for the present invention and the like. Reactions and purification techniques may be include, for example, immunoglobulins, cytokines, enzymes, performed e.g., using kits according to manufacturer's speci hormones, cancer antigens, nutritional markers, tissue spe fications, as commonly accomplished in the art or as cific antigens, and biowarfare agents. These protein analytes described herein. The foregoing techniques and procedures may be present in blood, serum, plasma, spinal fluid, synovial may be generally performed according to conventional meth fluid, saliva, urine, semen, prosthetic fluid, cells or tissues. ods well known in the art and as described in various general 0029. The following are examples of classes of protein or more specific references that are cited and discussed analytes related by structure that may be detected using the throughout the present specification. See e.g., Sambrooketat. compositions, methods and kits of the present invention: Molecular Cloning: A Laboratory Manual (2d ed., Cold 0030) protamines Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), Harlow & Lane, Antibodies: A Laboratory Manual 0031 histones (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 0032 albumins N.Y. (1988)), which are incorporated herein by reference in 0033 globulins their entirety for any purpose. 0034 scleroproteins US 2011 /023.0644 A1 Sep. 22, 2011

0035 phosphoproteins 0097 C7 0.036 mucoproteins 0.098 C'8 0037. The following examples are clinically important 0099 C'9 proteins found in human plasma that may be detected using 0100 Important blood clotting factors that may be the compositions, methods and kits of the present invention: detected using the compositions, methods and kits of the 0038 C-Lipoprotein present invention include the examples listed in the Table 0039 C.1-Antitrypsin below. 0040 Transcortin 0041 4.6S-Postalbumin TABLE 1 0042 Tryptophan-poor 0043 O-Glycoprotein BLOOD CLOTTING FACTORS 0044 al-Glycoprotein International 0045 Thyroxin-binding globulin Designation Name Inter-O-trypsin-inhibitor 0046) I Fibrinogen 0047 Gc-globulin II Prothrombin 0048 (Gc 1-1) IIa Thrombin 0049 (Gc 2-1) III Tissue thromboplastin V and VI Proaccelerin, accelerator globulin 0050 (Gc 2-2) VII Proconvertin 0051 Haptoglobin VIII Antihemophilic globulin (AHG) 0.052 (Hp 1-1) IX Christmas factor plasma thromboplastin component (PTC) 0053 (Hp 2-1) X Stuart-Prower factor, autoprothrombin III 0054 (Hp 2-2) XI Plasma thromboplastin 0055 Ceruloplasmin XIII Fibrin-stabilizing factor 0056 Cholinesterase 0.057 C-Lipoprotein(s) 0101 Important protein hormones that may be detected 0.058 Myoglobin using the compositions, methods and kits of the present 0059 C-Reactive Protein invention include: 0060 C-Macroglobulin 0102 Peptide and Protein Hormones 0061 C-HS-glycoprotein 0.103 Parathyroid hormone (parathromone) 0062 Zn-O-glycoprotein 0.104) Thyrocalcitonin 0063 C-Neuramino-glycoprotein 01.05 Insulin 0.064 Erythropoietin 0106 Glucagon 0065 B-lipoprotein 01.07 Relaxin 0066 Transferrin 0108) Erythropoietin 0067 Hemopexin 0.109 Melanotropin (melanocyte-stimulating hormone: 0068 Fibrinogen intermedin) 0069 Plasminogen 0110. Somatotropin (growth hormone) 0070 B-glycoprotein I 0.111 Corticotropin (adrenocorticotropic hormone) 0071 f-glycoprotein II 0112 Thyrotropin 0072 Immunoglobulin G 0113. Follicle-stimulating hormone 0073 (IgG) or YG-globulin 0114 Luteinizing hormone (interstitial cell-stimulating 0.074 Mol. formula: Y2k2 or Y272 hormone) 0075) Immunoglobulin A (IgA) or YA-globulin 0115 Luteomammotropic hormone (luteotropin, pro 0076 Mol. formula: (C. K.)" or (CK)" lactin 0.077 Immunoglobulin M (IgM) or YM-globulin 0116 Gonadotropin (chorionic gonadotropin) 0078 Mol. formula: (Ll, K2) or (Ll 2)s 0.117 Tissue Hormones 0079 Immunoglobulin D (Ig)) or YD-Globulin (YD) 0118 Secretin 0080 Mol. formula: (..delta. K) or .delta. K.) 0119 Gastrin 0081 Immunoglobulin E (IgE) or YE-Globulin (YE) 0120 Angiotensin I and II 0082 Mol. formula: (e. K) or (ew) 0121 Bradykinin 0083 Free K and w light chains 0.122 Human placental lactogen 0084 Complement factors: (0123 Cytokines 0085 C1 0.124 IL 1 0086 0.125 IL 2 0087 0.126 IL 4 0088 O127 IL 6 0089 C2 0128 II 8 0090 C3 0129 II 10 0091 C3 0.130 EGF 0092 BA 0131 TNF 0093 CD 0.132. NGF 0094) C4 0.133 Cancer Antigens 0.095 0134) PSA 0.096 0.135 CEA US 2011 /0230644 A1 Sep. 22, 2011

10136 C-fetoprotein (0195 Hemophilus-Bordetella Group 0.137 Acid phosphatase 0.196 Hemophilus influenza, 0138 CA19-9 (0197) Hemophilus ducryi 0139 CA125 0198 Hemophilus hemophilus 0140 Tissue Specific Antigens (0199 Hemophilus aegypticus 0141 alkaline phosphatase 0200 Hemophilus parainfluenza 0142 myoglobin 0201 Bordetalla pertussis 0143 CPK-MB 0202 Pasteurellae 0144) Troponin (0203 Pasteurella pestis (0145 BNP 0.146 Pro-BNP 0204 Pasteurella tulareusis 10147 Calcitonin 0205 Brucellae 0148 Myelin basic protein 0206 Brucella melitensis 0149 Peptide Hormones from the Neurohypophysis 0207 Brucella abortus 0150 Oxytocin 0208 Brucella suis 0151. Vasopressin 0209 Aerobic Spore-forming Bacilli 0152) Releasing factors (RF) CRF, LRF TRF, Soma 0210 Bacillus anthracis totropin-RF, GRF, FSH-RF, PIF, MIF 0211 Bacillus subtilis 0153 Ricin 0212 Bacillus megaterium 0154) Diptheria toxin 0213 Bacillus cereus 0155 Botulism toxin 0214) Anaerobic Spore-Forming Bacilli 0156) Staphylococcus enterotoxin B 0215 Clostridium botulinum 0157 Bacteria and viruses are also analytes that may be 0216 Clostridium tetani detected using the compositions, methods and kits of the 0217 Clostridium perfringens present invention. Included among these biological analytes 0218 Clostridium novyi are, among others: 0219) Clostridium septicum 0158 Corynebacteria 0220 Clostridium histolyticum 0159) Corynebacterium diphtheria 0221 Clostridium tertium (0160 Pneumococci 0222 Clostridium bifermentans (0161 Diplococcus pneumoniae 0223) Clostridium sporogenes (0162 Streptococci 0224 Mycobacteria (0163 Streptococcus pyrogens 0225. Mycobacterium tuberculosis hominis (0164. Streptococcus salivarus 0226 Mycobacterium bovis (0165 Staphylococci 0227. Mycobacterium avium (0166 Staphylococcus aureus 0228) Mycobacterium leprae (0167 Staphylococcus albus 0229 Mycobacterium paratuberculosis (0168 Neisseria 0230 Actinomycetes (fungus-like Bacteria) (0169. Neisseria meningitidis 0231 Actinomyces Isaeli (0170 Neisseria gonorrhea 0232 Actinomyces bovis (0171 Enterobacteriaciae 0233 Actinomyces naeslundii (0172 Coliform 0234 Nocardia asteroides (0173 Escherichia coli 0235 Nocardia brasiliensis (0174) Aerobacter aerogenes 0236). The Spirochetes (0175 Klebsiella pneumoniae 0237 Treponema pallidum (0176) Salmonellae 0238 Treponema pertenue 0.177 Salmonella typhosa 0239 Treponema carateum 0178 Salmonella choleraesuis 0240 Borrelia recurrentis 0179 Salmonella typhimurium 0241 Leptospira icterohemorrhagiae 0180 Shigellae 0242 Leptospira canicola 0181 Shigella dysenteria 0243 Trypanasomes 0182 Shigella schmitzii 0244 Mycoplasmas 0183) Shigella arabinotard 0245) Mycoplasma pneumoniae 0184 Shigella flexneri 0185 Shigella boydii 0246. Other Pathogens 0186. Shigella sonnei 0247 Rickettsiae (bacteria-like parasites) 0187. Other enteric bacilli 0248 Rickettsia prowazeki 0188 Proteus vulgaris 0249 Rickettsia mooseri (0189 Proteus mirabilis (0250 Rickettsia rickettsii (0190. Proteus species 0251 Rickettsia conori (0191) Proteus morgani 0252 Rickettsia australis (0192 Pseudomonas aeruginosa 0253 Rickettsia sibiricus 0.193) Alcaligenes faecalis 0254 Rickettsia akari 0.194 Vibrio cholerae 0255 Rickettsia tsutsugamushi US 2011 /023.0644 A1 Sep. 22, 2011

0256 Chlamydia (unclassifiable parasitesbacterial/vi 0315 Arboviruses ral) 0316 Eastern Equine Encephalitis Virus 0257 Chlamydia agents (naming uncertain) 0317 Western Equine Encephalitis Virus 0258 Fungi 0318. Sindbis Virus 0259 Cryptococcus neoformans 0319 Chikugunya Virus 0260 Blastomyces dermatidis 0320 Semliki Forest Virus 0261 Hisoplasma capsulatum 0321 Mayora Virus 0262 Coccidioides immitis 0322 St. Louis Encephalitis Virus 0263 Paracoccidioides brasiliensis 0323 Rickettsia prowaZeki 0264 Candida albicans 0324 California Encephalitis Virus 0265 Aspergillus fumigatus 0325 Colorado Tick Fever Virus 0266 Mucor corymbifer (Absidia corymbifera) 0326 Yellow Fever Virus 0267 Rhizopus Oryzae 0327 Dengue Virus 0268 Rhizopus arrhizua 0328. Reoviruses 0269 Phycomycetes 0329. Reovirus Types 1-3 0270 Rhizopus nigricans 0330. Retroviruses 0271 Sporotrichum schenkii 0331 Human Immunodeficiency Viruses I and II 0272 Flonsecaea pedrosoi (HIV) 0273 Fonsecacea compact 0332 Human T-cell Lymphotrophic Virus I & II 0274 Fonsecacea dermatidis (HTLV) 0275 Cladosporium carrionii 0333 Hepatitis 0276 Phialophora verrucose 0334 Hepatitis A Virus (0277 Aspergillus nidulans 0335 Hepatitis B Virus 0278 Madurella mycetomi 0336 Hepatitis C Virus 0279 Madurella grisea 0337 Tumor Viruses 0280 Allescheria boydii 0338 Rauscher Leukemia Virus (0281 Phialophora jeanselmei 0339 Gross Virus 0282 Microsporum gypseum 0340 Maloney Leukemia Virus 0283 Trichophyton mentagrophytes (0341 Human PapillomaVirus 0284 Keratinomyces aielloi 0342. In addition, it may be desirable to detect normal or 0285 Microsporum canis diseased tissue or cells of a patient. The presence or absence 0286 Microsporum adouini of certain circulating cancer or other cells, for example, may Trichophyton rubrum be diagnostic for disease. Thus, the endogenous cells of a 0287 human patient are analytes that may be advantageously Viruses detected using the compositions, methods and kits of the present invention. 0288 Adenoviruses 0343. The term “affinity molecule' as used herein refers to 0289 Herpes Viruses any molecule that is capable of specifically binding another 0290 Herpes simplex molecule. In one embodiment, the affinity molecule is an 0291 Varicella (Chickenpox) antibody. In another embodiment, the affinity molecule is an 0292 Herpes Zoster (Shingles) antigen. In other embodiments of the invention, affinity mol 0293 Virus B ecules can include, without limitation: nucleic acids (DNA, 0294 Cytomegalovirus RNA, PNA and nucleic acids that are mixtures thereof or that 0295) Pox Viruses include nucleotide derivatives or analogs); biological recep 0296 Variola (smallpox) tor molecules, such as the insulin receptor, ligands for recep 0297 Vaccinia tors (e.g., insulin for the insulin receptor); and biological, 0298 Poxvirus bovis chemical or other molecules that have affinity for another 0299 Paravaccinia molecule, such as biotin and avidin. The affinity molecules of Molluscum contagiosum the present invention need not comprise an entire naturally 0300 occurring molecule but may consist of only a portion, frag 0301 Picornaviruses ment or subunit of a naturally or non-naturally occurring 0302) Poliovirus molecule, as for example the Fab fragment of an antibody. O303 Coxsackievirus The affinity molecule may further comprise a marker that can 0304 Echoviruses be detected. 0305 Rhinoviruses 0344 Affinity molecules may be generated by any method (0306 Myxoviruses known in the art. For example, antibodies may be found in an 0307 Parainfluenza (1-4) antiserum, prepared from a hybridoma tissue culture Super 0308 Mumps Virus natant or ascites fluid, or may be derived from a recombinant 0309 Newcastle Disease Virus expression system, as will be well known in the art. Frag 0310 Measles Virus ments, portions or Subunits of e.g., an antibody, receptor or 0311 Rinderpest Virus other species, may be generated by chemical, enzymatic or 0312 Canine Distemper Virus other means, yielding for example, well-known (e.g., Fab, 0313 Respiratory Syncytial Virus Fab') or novel molecules. The present invention also contem 0314) Rubella Virus plates that affinity molecules can include recombinant, chi US 2011/023.0644 A1 Sep. 22, 2011

meric and hybrid molecules, such as humanized and prima producing the MAb of this invention may be cultivated in tized antibodies, and other non-naturally occurring antibody vitro or in vivo. Production of high titers of MAbs in vivo forms. Those skilled in the art will recognized that the non makes this a presently preferred method of production. limiting examples given above describing various forms of 0348. In addition, techniques developed for the production antibodies can also be extended to other affinity molecules of "chimeric antibodies' (Morrison, et al., Proc. Natl. Acad. Such that recombinant, chimeric, hybrid, truncated etc., forms Sci., 81:6851-6855 (1984); Takeda, etal, Nature, 314:452-54 of non-antibody molecules can be used in the methods of the (1985)) by splicing the genes from a mouse antibody mol present invention. ecule of appropriate antigen specificity together with genes 0345. By the terms “specifically binding and “specific from a human antibody molecule of appropriate biological binding as used herein is meant that an antibody or other activity can be used. A chimericantibody can be a molecule in molecule, especially an affinity molecule of the invention, which different portions are derived from different animal binds to a target Such as an antigen, ligand or other analyte, species, such as those having a variable region derived from a with greater affinity than it binds to other molecules under the murine MAb and a human immunoglobulin constant region. specified conditions of the present invention. Antibodies or 0349. Alternatively, techniques described for the produc antibody fragments, as known in the art, are polypeptide tion of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, molecules that contain regions that can bind other molecules, Science 242:423-26 (1988); Huston, et al. Proc. Natl. Acad. Such as antigens. In various embodiments of the invention, Sci. USA, 85:5879-83 (1988); and Ward, et al., Nature, 334: “specifically binding may mean that an antibody or other 544-46 (1989)) can be adapted to produce gene-single chain affinity molecule, binds to a target analyte molecule with at antibodies suitable for use in the present invention. Single least about a 10-fold greater affinity, preferably at least about chain antibodies are typically formed by linking the heavy a 10-fold greater affinity, more preferably at least about a and light chain fragments of the FV region via an amino acid 10-fold greater affinity, and most preferably at least about a bridge, resulting in a single chain polypeptide. 10-fold greater affinity than it binds molecules unrelated to 0350 Antibody fragments that recognize specific epitopes the target molecule. Typically, specific binding refers to may be generated by known techniques. For example, Such affinities in the range of about 10-fold to about 10-fold fragments include but are not limited to: the F(ab')2 fragments greater than non-specific binding. In some embodiments, spe that can be produced by pepsin digestion of the antibody cific binding may be characterized by affinities greater than molecule and the Fab fragments that can be generated by 10-fold over non-specific binding. Whenever a range reducing the disulfide bridges of the F(ab'), fragments. Alter appears herein, as in 1-10 or one to ten, the range refers natively, Fab expression libraries may be constructed (Huse, without limitation to each integer or unit of measure in the et al., Science, 246:1275-81 (1989)) to allow rapid and easy given range. Thus, by 1-10 it is meant each of 1, 2, 3, 4, 5, 6, identification of monoclonal Fab fragments with the desired 7, 8, 9, 10 and any subunit in between. specificity. (0346 “Polyclonal Antibodies” or “PAbs,” are heteroge 0351. The term “hapten as used herein, refers to a small neous populations of antibody molecules derived from the proteinaceous or non-protein antigenic determinant which is sera of animals immunized with an antigen, or an antigenic capable of being recognized by an antibody. Typically, hap functional derivative thereof. For the production of poly tens do not elicit antibody formation in an animal unless part clonal antibodies, host animals such as rabbits, mice and of a larger species. For example, Small peptide haptens are goats, may be immunized by injection with an antigen or frequently coupled to a carrier protein such as keyhole limpet hapten-carrier conjugate, optionally Supplemented with adju hemocyanin in order to generate an anti-hapten antibody vants. Polyclonal antibodies may be unpurified, purified or response. "Antigens' are macromolecules capable of gener partially purified from other species in an antiserum. Tech ating an antibody response in an animal and being recognized niques for the preparation and purification of polyclonal anti by the resulting antibody. Both antigens and haptens com bodies are well-known in the art and are described in various prise at least one antigenic determinant or "epitope, which is general and more specific references, including but not lim the region of the antigen or hapten which binds to the anti ited to Kabat & Mayer, Experimental Immunochemistry, 2d body. Typically, the epitope on a hapten is the entire molecule. ed., (Thomas, Springfield, Ill. (1961)); Harlow & Lane, Anti 0352 “Receptor” or “biological receptor” typically refers bodies: A Laboratory Manual (Cold Spring Harbor Labora to a molecular structure within or on the surface a cell char tory Press, Cold Spring Harbor, N.Y. (1988)); and Weir, acterized by selective binding of a specific Substance (e.g. a Handbook of Experimental Immunology, 5th ed. (Blackwell “ligand) and resulting in a specific physiologic effect that Science, Cambridge, Mass. (1996)). accompanies the binding. Examples of receptors include cell 0347 “Monoclonal antibodies, or “blabs”, which are Surface receptors for peptide hormones, neurotransmitters, homogeneous populations of antibodies to a particular anti antigens, complement fragments and immunoglobulins and gen, may be obtained by any technique that provides for the cytoplasmic receptors for steroid hormones. As used herein, production of antibody molecules, such as by continuous however, the receptor will typically be isolated and purified culture of cell lines. These techniques include, but are not and need not effect or be capable of effecting a physiological limited to the hybridoma technique of Köhler and Milstein, or other biological effect. The methods of the present inven Nature, 256:495-7 (1975); and U.S. Pat. No. 4,376,110), the tion exploit the selective binding of the receptor to the specific human B-cell hybridoma technique (Kosbor, et al., Immunol Substance. ogy Today, 4:72 (1983); Cote, et al., Proc. Natl. Acad. Sci. 0353. The term “ligand’ refers generally to a molecule USA, 80:2026-30 (1983)), and the EBV-hybridoma technique that binds to a receptor. Typically, a ligand is a small, Soluble (Cole, et al., in Monoclonal Antibodies And Cancer Therapy, molecule. Such as a hormone or neurotransmitter. Alan R. Liss, Inc., New York, pp. 77-96 (1985)). Such anti 0354. The term "solid support” refers any solid phase that bodies may be of any immunoglobulin class including IgG, can be used to immobilize e.g., an analyte, an antibody or a IgM, IgE, IgA, Ig|D and any Subclass thereof. The hybridoma complex. Suitable solid supports will be well known in the art US 2011/023.0644 A1 Sep. 22, 2011

and include the walls of wells of a reaction tray, Such as a 500 amino acids. “Proteins include single polypeptides as microtiter plate, the walls of test tubes, polystyrene beads, well as complexes of multiple polypeptide chains, which may paramagnetic or non-magnetic beads, nitrocellulose mem be the same or different. Multiple chains in a protein may be branes, nylon membranes, microparticles such as latex par characterized by secondary, tertiary and quaternary structure ticles, and sheep (or other animal) red blood cells. Typical as well as the primary amino acid sequence structure; may be materials for Solid Supports include, but are not limited to, held together, for example, by disulfide bonds; and may polyvinyl chloride (PVC), polystyrene, cellulose, nylon, include post-synthetic modifications such as, without limita latex and derivatives thereof. Further, the solid support may tion, glycosylation, phosphorylation, truncations or other be coated, derivatized or otherwise modified to promote adhe processing. Antibodies such as IgG proteins, for example, are sion of the desired molecules (e.g., analytes) and/or to deter typically comprised of four polypeptide chains (i.e., two non-specific binding or other undesired interactions. The heavy and two light chains) that are held together by disulfide choice of a specific “solid phase' is usually not critical and bonds. Furthermore, proteins may include additional compo can be selected by one skilled in the art depending on the nents such as associated metals (e.g., iron, copper and Sulfur), assay employed. Thus, latex particles, microparticles, para or other moieties. The definitions of peptides, polypeptides magnetic or non-magnetic beads, membranes, plastic tubes, and proteins include, without limitation, biologically active walls of microtiter wells, glass or silicon chips, and red blood cells are all suitable sold supports. Conveniently, the solid and inactive forms; denatured and native forms; as well as Support can be selected to accommodate various detection variant, modified, truncated, hybrid, and chimeric forms methods. For example, 96 or 384 well plates can be used for thereof. The peptides, polypeptides and proteins of the assays that will be automated, for example by robotic work present invention may be derived from any source or by any stations, and/or those that will be detected using, for example, method, including, but not limited to extraction from natu a plate reader. For methods of the present invention that may rally occurring tissues or other materials; recombinant pro involve an autoradiographic or chemiluminescent detection duction in host organisms such as bacteria, fungi, plant, insect step utilizing a film-based visualization, the Solid Support or animal cells; and chemical synthesis using methods that may be a thin membrane, such as a nitrocellulose or nylon will be well known to the skilled artisan. membrane. According to one embodiment of the invention in 0356. The term “conjugate' as used herein refers to two which sandwich immunoassays are performed, the walls of molecules that have been covalently attached, or otherwise the wells of a reaction tray are typically employed. In alter linked together. In one embodiment, a nucleic acid conjugate native embodiments of the instant invention, paramagnetic is generated by covalently linking a nucleic acid to a protein, beads may be used as a solid support. Suitable methods for polypeptide or other affinity molecule. In a preferred embodi immobilizing molecules on Solid phases include ionic, hydro ment of the invention, the protein, polypeptide or other affin phobic, covalent interactions and the like, and combinations ity molecule is covalently attached to a nucleic acid via a thereof. However, the method of immobilization is not typi linking group to form a conjugate. cally important, and may involve uncharacterized adsorption 0357 A“kit' for detecting the presence of an analyte in a mechanisms. A “solid Support’ as used herein, may thus refer sample by the methods of the invention may, by way of to any material which is insoluble, or can be made insoluble example, comprise at least one container means having dis by a Subsequent reaction. The solid Support can be chosen for posed therein a binding pair specific for the selected analyte. its intrinsic ability to attract and immobilize a capture reagent. The kit may further comprise other containers comprising Alternatively, the Solid phase can retain an additional receptor one or more of the following: buffers, solutions or other which has the ability to attract and immobilize a capture reagents and materials necessary for performing analyte reagent. The additional receptor may include a Substance that detection; reagents capable of amplifying the nucleic acid is oppositely charged with respect to either the capture probe components of the binding pairs; and reagents capable reagent itself or to a charged substance conjugated to the of detecting the presence of nucleic acid components follow capture reagent. In yet another embodiment of the invention, ing amplification. Preferably, the kit further comprises an additional receptor molecule can be any specific binding instructions for use. The kit, if intended for diagnostic use, member which is immobilized upon (attached to) the solid may also include notification of a FDA approved use and phase and which has the ability to immobilize a capture instructions therefor. reagent through a specific binding reaction. The additional 0358 Specifically, a compartmentalized kit includes any receptor molecule enables indirect immobilization of the cap kit in which reagents are contained in separate containers. ture reagent to a solid phase before or during the performance Such containers include Small glass containers, plastic con of the assay. The Solid phase thus can be a plastic, derivatized tainers or strips of plastic or paper. Such containers allow the plastic, paramagnetic or non-magnetic metal, glass or silicon efficient transfer of reagents from one compartment to surface of a test tube, microtiter well, sheet, bead, micropar another compartment Such that the samples and reagents are ticle, chip, or other configurations known to those of ordinary not cross-contaminated and the agents or Solutions of each skill in the art. container can be added in a quantitative fashion from one 0355 “Peptide’ generally refers to a short chain of amino compartment to another. Such containers may include a con acids linked by peptide bonds. Typically peptides comprise tainer which will accept a test sample, a container which amino acid chains of about 2-100, more typically about 4-50, contains the probe or primers used in the assay, containers and most commonly about 6-20 amino acids. “Polypeptide' which contain buffers and reagents (such as phosphate buff generally refers to individual Straight or branched chain ered saline, Tris-buffers, and the like), and containers which sequences of amino acids that are typically longer than pep contain the reagents used to detect the marker nucleic acid, tides. “Polypeptides' usually comprise at least about 100 to amplified product, or the like. One skilled in the art will 1000 amino acids in length, more typically at least about 150 readily recognize that preformed binding pairs and/or mate to 600 amino acids, and frequently at least about 200 to about rials, Supplies and reagents necessary to prepare binding pairs US 2011/023.0644 A1 Sep. 22, 2011

can readily be incorporated into one of the established kit 0363 The protein microbubbles of the invention are typi formats that are well known in the art. cally formed by the introduction of a gas into a solution of 0359 A kit for coupling DNA to an antibody or other protein, for example, by sonication. The bubbles can be filled affinity molecule by the methods of the invention may com with any gas, including, but not limited to oxygen, nitrogen, prise at least one container means having disposed therein the carbon dioxide, helium, fluorocarbon gases and various com binations thereof. Such as air. In one aspect of the invention, lyophilized activated DNA. The kit may further comprise gas may be introduced into the protein through a process other containers comprising one or more of the following: comprising heating a solution of protein. Without being lim reagents, buffers and agents capable of detecting the presence ited to a specific theory, heating may serve to stabilize the of nucleic acid after the reaction. Preferably, the kit further protein microbubbles by denaturing the protein. In another comprises instructions for use. A kit for making or using the embodiment, the protein microbubbles may be stabilized, for novel compositions and methods for isolating and assaying example, by denaturing the protein, fixing the protein, analytes described herein may include the already manufac crosslinking the protein or treatment with Cr". In one tured microbubble or the thin film used to create the aspect, the microbubbles are stabilized by cross linking with microbubble. Likewise, the microbubble of the kit may or aldehydes such as glutaraldehyde or formaldehyde. may not already have the attached affinity molecule. The kit 0364 Protein microbubbles are generally in the range of may further comprise other containers comprising one or 0.1 to 100 microns, typically 1 to 50, and frequently 2 to 20 or more of the following: reagents, buffers and agents capable of 2 to 30 microns in diameter. making the microbubbles or useful in employing them for 0365 According to the invention, the affinity molecule methods of affinity isolation, purification, concentration, etc. can be a receptor, a ligand, or an antibody. Alternatively, the Preferably, the kit further comprises instructions for use. One affinity molecule can be biotin, avidin or streptavidin. In one skilled in the art will readily recognize that compositions and embodiment of the invention, the microbubbles are biotiny methods described in the present invention can readily be lated and then coated with streptavidin, which creates a incorporated into one of the established kit formats that are microbubble that can be readily coated with a biotinylated well known in the art. ligand Such as an antibody. In another embodiment, the 0360. The present invention provides novel compositions microbubbles bubbles are ligand-coated microbubbles. and methods for isolating and assaying analytes, including 0366 Coating of microbubbles with an affinity molecule also cells, viruses, cellular Subcomponents and soluble mol can be accomplished by any method known in the art. Advan ecules from solution. It relies on coated microbubbles to tageously, proteins contain amine functional groups that can specifically bind the target analyte (cell, virus, cellular Sub serve as the basis for numerous modifications and coupling component or soluble molecule). According to the invention, reaction, such as reaction with aldehydes. Furthermore, the microbubbles are coated with, or otherwise made to exhibit skilled artisan will recognize that the materials that make up on their exterior surface, an affinity molecule. The novel microbubbles, e.g. protein, glass and the like, can be chemi compositions and methods of the present invention can also cally derivatized or functionalized to covalently interact with be used to concentrate analytes, including but not limited to various types of affinity molecules. The skilled artisan will be antibodies, antigens, proteins and nucleic acids. familiar with a variety of commercial reagents, products and 0361. In one embodiment, the affinity-molecule coated kits for coupling proteins and other molecules to the microbubbles further comprise a detectable marker. In still microbubbles of the invention. The affinity molecule may be another embodiment, the affinity molecule itself is the detect directly coupled to the protein, for example, by using a het able marker. In these embodiments, the amount, or present or erobifunctional reagent Such as Sulfo Succinimidyl 4-(N-ma absence of the analyte, species, or microbubble may be quan leimidomethyl)cyclohexane-1-carboxylate. tified or detected by virtue of the marker. In one embodiment, 0367. In another embodiment of the invention, the affinity the marker on the microbubble is a nucleic acid that may be molecule is indirectly coupled to the protein, Such as through amplified and detected. The techniques used to accomplish the interaction of at least one other molecule. In one aspect of detection may include, but are not limited to, PCR, nucleotide the invention, microbubbles are directly coupled to streptavi sequencing, PCR sequencing, molecular beacon technology, din and the affinity molecule is biotinylated, such that the hybridization, hybridization followed by PCR, fluorescence, streptavidin and biotin interact to couple the affinity molecule radiolabelling, phosphorescence and absorbance. Examples to the protein. The interactions of biotin and avidin/strepta of reagents that may be used for detection include, but are not vidin are well known in the art, as are methods for coupling limited to, radiolabels, enzymatic labels (e.g. horseradish these molecules to other species. Reference may also be made peroxidase, alkaline phosphatase), fluorescence, phosphores to a general or more specific textbook or laboratory manual cence, bioluminescence, chemiluminescence, affinity labels describing the chemistry, biology, and interactions of biotin, (e.g. biotin, avidin, or streptavidin) and other reagents well avidin and Streptavidin, and/or the methods for coupling known by those of skill in the art. These embodiments are not biotin and avidin/streptavidin to other molecules. See e.g., limiting, and other embodiments can be envisioned being Avidin-Biotin Chemistry: A Handbook (Savage, et. al., eds. used with the invention. Pierce Chemical Co., Rockford, Ill., 1992) 0362. In one embodiment of the invention, the 0368. In another aspect of the invention, coated microbubbles are protein microbubbles, which can be com microbubbles are made using glass microbubbles Such as prised of any peptide, polypeptide, protein or combinations those supplied by 3MTM. Borosilicate glass bubbles can be thereof. Both synthetic and naturally occurring peptides, treated with sodium hydroxide to expose a silica Surface and polypeptides, proteins and combinations are contemplated by then reacted with a silanating agent such as an 3-amino the invention. In one embodiment, the protein microbubbles propyl-triethoxy silane, creating a surface coated with a pri can be readily formed into microbubbles through the intro mary amines. The microbubble can be reacted with NHS duction of a gas. In one embodiment, the protein is albumin. biotin to form a biotinylated glass microbubble, which can US 2011/023.0644 A1 Sep. 22, 2011 then be coated with streptavidin, if desired. This streptavidin factant. This aspect of the invention is particularly useful microbubble can then be easily coated with a biotinylated where it is desirable to isolate the target species devoid of the ligand Such as a biotinylated antibody; alternatively epoxy capturing microbubble. For example, it may be desirable to coated glass micro bubbles can be reacted directly with characterize the phenotype of an affinity-isolated cell or to ligands or coated directly or indirectly by methods known to free the isolated cell for further analysis or propagation. The those skilled in the art. methods of the present invention provide, in some embodi 0369. In another embodiment of the invention, methods ments, a simple means for releasing the species from the for affinity isolation or affinity assay of a species are provided. capturing microbubble that avoids potentially damaging According to these embodiments, the method comprises the reagents, such as enzymes, harsh chemicals and extremes of steps of providing microbubbles coated with an affinity mol pH. In other embodiments, the analyte or species may be ecule in a solution, contacting the microbubbles with a spe released from the microbubble by enzymatic or chemical cies that interacts with the affinity molecule in a solution, CaS. thereby generating microbubbles coated with the species, and 0374 Glass microbubbles advantageously can be con separating the microbubbles coated with the species from the structed from borosilicate glass which is largely free of con Solution in a preferred embodiment, allowing the taminating material. This material is also resistant to break microbubbles coated with the species to float to the top of the age or destruction during normal handling. Solution—thereby separating the species from the solution. In 0375. The microbubbles of the present invention have an this manner, all manner of species may be affinity isolated or additional advantage over Solid particles for affinity applica affinity assayed including proteins (antigen, antibodies, tions in that in the normal force of gravity and the buoyant ligands, receptors, hormones), nucleic acids, lipoproteins, force of the microbubble are in different directions, thus fats, triglycerides, Sugars, carbohydrates, viruses, cells, cel resulting in a significant reduction in non specific binding and lular components, Subcellular organelles, and components of entrapment of species that typically sink toward the bottom of Subcellular organelles, as well as complexes thereof the reaction vessel during separation. The separation can be 0370. In yet another embodiment of the invention, meth enhanced with the unbound cells being forced away from the ods for affinity concentration of a species are provided. microbubbles in a low centrifugal field, as with a modest According to these embodiments, the method comprises the centrifugal speed, under conditions that do not adversely steps of providing microbubbles coated with an affinity mol affect the microbubbles. ecule in a solution, contacting the microbubbles with a spe 0376. As described further below under EXAMPLES, cies that interacts with the affinity molecule in a solution, experiments with albumin microbubbles coated with an anti thereby generating microbubbles coated with the species, and bacteria antibody mixed with a suspension of bacteria have separating the microbubbles coated with the species from the resulted insterilization of the suspension, with all the bacteria Solution in a preferred embodiment, allowing the co-isolated with the microbubbles. Similarly, in control microbubbles coated with the species to float to the top of the experiments the microbubbles without specific antibody Solution—thereby separating the species from the solution. In showed little non-specific binding, resulting in no detectable this manner, all manner of species may be concentrated Such bacteria co-purifying with the microbubbles following sepa as proteins (antigen, antibodies, ligands, receptors, hor ration. mones), nucleic acids, lipoproteins, fats, triglycerides, Sug 0377 The present invention also provides methods for ars, carbohydrates, viruses, cells, cellular components, Sub generating microbubbles for use in affinity isolation or affin cellular organelles, and components of Subcellular ity assay comprising providing microbubbles; and coating the organelles, as well as complexes thereof. microbubbles with an affinity molecule. 0371. The species that can be isolated, assayed, purified or 0378. It will be understood that the application of the concentrated can be any manner of material including pro teachings of the present invention to a specific problem or teins (antigen, antibodies, ligands, receptors, hormones), situation will be within the capabilities of one having ordinary nucleic acids (RNA, DNA nucleotide analogs, mixtures skill in the art in light of the teachings contained herein. The thereof, etc), lipoproteins, fats, triglycerides, Sugars, carbo invention will be further illustrated by reference to the fol hydrates, viruses, cells, cellular components (liposomes, lowing non-limiting Examples. The following examples, endoplasmic reticulum, etc.). Subcellular organelles (mito including experiments and results achieved, are provided for chondria, etc.), and components of Subcellular organelles, as illustrative purposes only and are not to be construed as lim well as complexes thereof. In one aspect of this embodiment, iting the present invention. the species is a receptor, a ligand, or an antigen. In one embodiment, the species is an analyte. In alternative embodi EXAMPLES ments, the species is a virus or a cell. Albumin Microbubbles 0372. The coated microbubbles of the invention bind to the target species, including cells, viruses, analytes or other Example 1 molecules and then rise to the surface of the solution, thus separating themselves from the contacting Solution and non Preparation of Albumin Microbubbles target species. In certain embodiments, where the separation 0379 Albumin solution (Human) 5%, USP (Bayer Cor time is important, the solution containing the microbubbles poration, Elkhart, Ind.) was diluted to 1% with air-saturated may be centrifuged or subjected to a bubble trap to further normal saline at room temperature. Twenty milliliters of the effect the separation more rapidly. diluted solution was placed into a 50 ml glass beaker and 0373 The albumin microbubbles of the invention have the immersed in a 85°C. water bath below the 25 ml level on the useful property of being able to be easily destroyed and made beaker. The temperature of the albumin solution was moni to visually disappear by applying pressure or vacuum to the tored using digital thermometer with gentle stirring of the Solution, or by adding a small amount of a detergent or Sur solution. At a process temperature of 73°C., the probe of a US 2011/023.0644 A1 Sep. 22, 2011

Branson Digital Sonifier(R), Model 450 (Branson Ultrasonics microbubbles were stored under refrigeration in PVA/PBS Corp., Danbury, Conn.) was placed in contact with the Surface containing 0.05% sodium azide. of the albumin solution and immediately sonicated at 80% amplitude for 10 seconds. The beaker was removed from the Example 4 water bath, placed in crushed ice, and stirred gently until the temperature was reduced to 40°C. The albumin microbubble Preparation of Streptavidin-Coated Albumin suspension was transferred to a 150 ml flexible carboy (Flex Microbubbles boy(R) Bag, Stedim, Concord, Calif.) at room temperature. 0382 Air-filled albumin microbubbles were coated with The process was repeated several times with fresh solution streptavidin (Prozyme, San Leandro, Calif.) indirectly by until the bag was filled. The microbubble suspension was exposing biotin-microbubbles to an excess of streptavidin. adjusted to 0.05% sodium azide and the bag stored vertically Under these conditions, cross-linking of microbubbles is under refrigeration for at least 24 hours. The Sonication pro avoided. Streptavidin was also coated onto albumin cess converted approximately 5% of the soluble albumin to microbubbles directly via a bifunctional protein cross-linking insoluble, air-filled albumin microbubbles. reagent. 0383 For indirect coating, a suspension of biotin-coated Example 2 albumin microbubbles suspended in PVA/PBS was treated Preparation of Chromium-Stabilized Albumin completely and rapidly by addition of 10 mg/ml solution of Microbubbles streptavidin to yield a final concentration of 1 mg/ml, with continuous Vortex mixing. Unreacted Streptavidin was 0380. In some experiments, albumin microbubbles were removed by repeated centrifugal washing as described above. stabilized by treatment with Cr". Albumin microbubbles The tetravalent nature of streptavidin ensured that biotin were allowed to float and the liquid phase was removed and binding sites were still available. replaced with 5 mM chromium potassium sulfate. The 0384 Alternatively, albumin microbubbles were coated microbubbles were maintained in Suspension by gentle agi directly with streptavidin using sulfosuceinimidyl 4-(N-ma tation and incubated in a water bath at 60° C. for 30 minutes. leimidomethyl)cyclohexane-1-carboxylate (s-SMCC). Following incubation, the chromium solution was removed Streptavidin at 10 mg/ml in PBS, pH 7.4, was treated with a 5 by washing as described below, and replaced with 1% human to 10-fold molar excess of n-succinimidyl S-acetylthioacetate serum albumin in normal saline. Chromium treated albumin (SATA) for at least 1 hour at room temperature. Unreacted microbubbles were treated as described for untreated SATA was removed from the streptavidin by FPLC using a microbubbles. Sephadex G-25 resin equilibrated in PBS, and the purified protein was stored frozen. Just prior to use, the protective Example 3 acetyl group was removed from the modified streptavidin by treatment with PBS containing 50 mM hydroxylamine and Preparation of Biotin-Coated Albumin Microbubbles 2.5 mM EDTA, pH 7.5, for 2 hours at room temperature. 0381 Unconverted albumin solution was drained away Simultaneously, a Suspension of albumin microbubbles Sus from underneath the floating layer of microbubbles and pended in PVA/PBS was treated with 0.01 to 1 mg/ml replaced by cold, air-saturated phosphate buffered saline, pH s-SMCC under gentle agitation for 30 min at room tempera 7.4 (PBS), containing 0.2% polyvinyl alcohol (MW 30,000 ture. Excess s-SMCC reagent was immediately removed by 70,000) (PVA/PBS). The microbubbles were resuspended by centrifugal washing (repeated 4 times), and the s-SMCC gentle agitation and transferred to disposable plastic syringes microbubbles were combined with the sulfhydryl-modified equipped with a bottom-mounted Stopcock. The streptavidin. The streptavidinbecomes covalently coupled to microbubbles were washed free of residual soluble albumin the surface of the albumin microbubbles by reaction of the by repeated centrifugation at 200xg, at 4° C. for 5 min, maleimide functional group with the freshly exposed strepta draining, and replenisment of the Solution with fresh cold, vidin sulfhydryl group. The microbubbles were washed free air-saturated PVA/PBS. The washed albumin microbubbles of excess streptavidin by repeated centrifugal washings, Sus were suspended at approximately 25% V/v in PVA/PBS and pended in cold, air-saturated PBS containing 0.05% sodium biotinylated by reaction with sulfosuccinimidyl-6-(biotina azide and stored under refrigeration. mido) hexanoate (sulfo-NHS-LC-biotin; BZ-LinkTM sulfo NHS-LC-biotin, Pierce Biotechnology, Inc., Rockford, Ill.) at Example 5 a concentration of 0.01 to 1.0 mg/ml under gentle agitation at Coating Albumin Microbubbles with Antibody room temperature for at least one hour. Unreacted biotin was removed by several centrifugal washings with cold, air-satu (0385 Affinity-purified antibody to E. coli O157:H7 was rated PVA/PBS at 4° C. The extent of biotin labeling was obtained from KPL (Gaithersburg, Md.) and biotinylated as assessed by dissolving an aliquot of Suspended microbubbles described below. The antibody was dissolved in PBS, pH 7.4 in PVA/PBS containing 0.1% Triton X-100 and determining and warmed to 37° C. for 30 min. The warmed solution was the concentrations of protein (BCA Protein Assay, Pierce) treated with an 8-12 fold molar excess of sulfosuccinimidyl and biotin (2-(4'-hydroxyazobenzene)-benzoic acid assay). 6-(biotinamido) hexanoate (EZ-LinkTM sulfo-NHS-LC-bi Typical biotinylation reactions of albumin microbubbles otin) for 2 hrs at room temperature. Excess biotin reagent was yield molar ratios of 40% to 500% biotin to albumin. The removed by G-25 Sephadex gel filtration chromatography, availability of biotin on the surface of the albumin eluting with PBS. The biotinylated antibody was concen microbubbles was confirmed by observing the spontaneous trated by ultra-filtration using a Microcon R YM-30 Filter association of biotin-microbubbles with BioMag Nuclease Device (Millipore, Bedford, Mass.) and stored under refrig Free Streptavidin paramagnetic particles (Polysciences, Inc., eration in the presence of sodium azide as preservative. Avi Warrington, Pa.) in suspension. The biotin-coated albumin din-coated albumin microbubbles were resuspended by US 2011/023.0644 A1 Sep. 22, 2011

gentle agitation and combined with a molar excess of biotin albumin were reactive with avidin or streptavidin and could labeled antibody in PBS. Excess material was removed from be coated with avidin or streptavidin as described above. the insoluble microbubbles by repeated centrifugation and resuspension as described above. The antibody-coated albu Glass Microbubbles min microbubbles were stored in PVA/PBS containing Example 8 sodium azide, at 4°C. Preparation of Amine-Coated Glass Microbubbles Example 6 0388 3MTM ScotchLiteTM Glass Bubbles S60HS (St Paul, Minn.), having a density of about 0.6 g/cc and an average Capture of Bacterial Cells on Antibody-Coated diameter of about 30 um, were suspended in water and Microbubbles allowed to float to the top of the suspension. The liquid layer was drained from the bottom to remove fines and shards. This was conveniently performed in a 60 cc disposable Syringe 0386 E. coil 0157:H7 was obtained from AmericanType fitted with a stopcock mounted on the tip. The washing was Culture Collection (Manassas, Va.) and propagated on Luria repeated several times. Reactive surface hydroxyl residues Bertani (LB) liquid and agar media at 37° C. Bacteria grown were provided by suspension of the washed microbubbles in in liquid media were serially diluted in cold, sterile PVA/PBS 0.25 MNaOH for 24 hours at 60° C., followed by washing or PBS containing 0.2% BSA (BSA/PBS) to a density of with water to remove the alkali. The microbubbles were sub approximately 5,000 cells/ml. One-tenth volume of sequently treated with 0.05 M HCl for 1 hour at room tem microbubble Suspension was added to the cell Suspension and perature. The acid was removed by washing with water, fol gently agitated for several minutes at room temperature. The lowed by dry acetone, and finally the product was dried in an microbubbles were allowed to float to the surface of the oven at 60° C. The hydroxyl functions were converted to mixture by natural buoyancy over 10 minutes. Ten microliters amino groups by Suspending the treated, dried glass of the underlying liquid phase, cleared of floating microbubbles in a 3% solution of 3-aminopropyltriethoxy microbubbles, was removed by micropipette, streaked onto silane (3-APS, Sigma) in dry acetone or toluene, for 30 min an LB agar plate and incubated overnight at 37° C. Positive utes at room temperature. Excess silane was removed by control samples of bacterial Suspension yielded approxi several washings with acetone, and the derivatized mately 50 colonies/plate. Bacterial suspension treated with microbubbles were oven dried at 60° C. antibody-coated microbubbles were depleted of colony form 0389. In some experiments, the glass microbubbles were ing cells. The bacterial cells could be recovered from the suspended in 70% sulfuric acid and 9% hydrogen peroxide microbubbles by gently agitating the microbubble Suspension for 16 hours at room temperature, followed by exhaustive and plating 10ul of the Suspension, as evidenced by colonies washing with water. The acid-treated microbubbles were formed on the resulting LB plates after overnight incubation. resuspended in a solution of 3-APS:water:ethanol (3:4:92 by Volume) prepared just prior to use. The reaction was allowed This result indicates that bacterial cells are captured by anti to proceed for 30 minutes at room temperature. Excess body-coated albumin microbubbles. Bacterial suspensions reagent was removed by washing in ethanol, and the glass treated with either uncoated or streptavidin-coated albumin microbubbles baked at 115° C. for 1 hour. The microbubbles microbubbles failed to remove cells from suspension. were washed again in ethanol and oven dried at 60° C. 0390 Amine-coated glass microbubbles were stored dry Example 7 at room temperature. The presence of functional Surface amine groups at 2.5-3.0/nm was confirmed by analysis using Preparation of Microbubbles By First Modifying s-succinimidyl-4-O-(4,4'dimethoxytrityl)butyrate (Pierce). Albumin Followed by Sonication Example 9 0387 Albumin microbubbles prepared from biotinylated Coating Amine-Glass Microbubbles with Antibody serum albumin by labeling the serum albumin with biotin prior to microbubble formation, were found to bind avidin 0391 Amine-coated glass microbubbles were suspended and/or streptavidin. Bovine Serum Albumin (BSA: Bovumi in 50 mM sodium bicarbonate, 0.1% TweenTM 20, pH 8.5, and nar Cohn Fraction V, intergen, Purchase, N.Y.) was dissolved reacted with sulfo-NHS-LC-biotin for two hours at room in normal saline, containing 4 mM sodium caprylate and 4 temperature. The biotin reagent was present at a 0.1 to 7-fold mM sodium tryptophanate to 50 mg/ml, sterile filtered and excess over available surface amines. The glass microbubbles stored in a clear glass container, under fluorescent lighting at were washed and stored in PBS containing 0.1% TweenTM20 room temperature for two weeks. This treatment photo-oxi (PBS/Tween)TM. Avidin, dissolved in water at 5 mg/ml, was dized free sulfhydryl groups that can interfere with added in molar excess to saturate the available biotin sites. microbubble formation. Twenty milliliters of the BSA solu Excess avidin was removed by washing with PBS/TweenTM tion was removed and adjusted to pH 8.5 with 1 M NaOH. and the microbubbles were stored in PBS, containing 0.2% Twenty-five micrograms of s-NHS-LC-biotin was added BSA (BSA/PBS) and 0.05% sodium azide, under refrigera with mixing and the pH maintained at 8.5 for 1 hour at room tion. temperature. The reaction was diluted to 1% albumin by the 0392 Amine glass microbubbles were also coated with addition of normal saline and Subjected to the Sonication sulfhydryl-modified avidin via s-SMCC cross-linking process as described above for unmodified human albumin. reagent. Amine glass microbubbles were suspended 1:10 The resulting albumin microbubbles were washed several (w/v) in 50 mM sodium phosphate, pH 7.5 containing 10% times with 1% BSA in normal saline to remove unincorpo dimethyl formamide and 2 mg/ml s-SMCC for one hour at rated biotin. The microbubbles prepared from biotinylated room temperature. The maleimide-coated glass bubbles were US 2011/023.0644 A1 Sep. 22, 2011

washed with 100% dry ethanol, drained, dried under vacuum dried overnight at 37° C. The cis-diol glass microbubbles in a glass vial, and stored under nitrogenat -20°C. An excess were stored dry at room temperature. amount of avidin, modified with SATA to contain free sulf 0397. A 25% suspension of cis-diol-coated glass hydryl groups (see Example 4 above), was deacetylated with microbubbles was activated with 0.3 M carbonyl diimidizole hydroxylamine and added directly to the dried maleimide (CDI) in dry acetone for 1 hour at room temperature in a glass microbubbles to yield avidin-coated microbubbles. sealed vessel, with venting every 10 minutes. Excess reagent Excess avidin was removed after overnight incubation by was removed by washing with dry acetone and the glass washing with PBS/TweenTM. microbubbles were dried under vacuum at room temperature. 0393 Glass microbubbles (20 mg) coated with avidin The activated microbubbles were stored dry under nitrogen at were suspended in 1.0 ml PBS/BSA and combined with 10 ug 40 C. of antibody to E. coli O157:H7, modified to contain 2-6 biotin 0398 CDI-activated glass microbubbles were coated by groups (see Example 5 above). The antibody readily attached overnight incubation with 4 mg/ml avidin dissolved in 0.1 M to the avidin Surface coating within two hours in an ice bath, sodium carbonate, pH 9.5 at room temperature. The pH was creating antibody-coated glass microbubbles. These reduced to 6.5 by the addition of 0.2 M sodium phosphate, microbubbles were washed and stored at 4°C. as a suspension monobasic. The avidin-coated glass microbubbles were in an aqueous buffer Suitable for maintaining antibody stabil washed and stored in BSA/PBS containing sodium azide. ity. BSA and sodium azide were added as stabilizers. 0399 Biotin-labeled antibody to E. coli O157:H7 was applied and bacteria removed from Suspension as previously Example 10 described above. This experiment gave a 50-100% reduction Capture of E. Coil 0157 Onto Antibody-Coated in bacteria, as evidenced by a reduced number of colonies Glass Microbubbles relative to control samples. 0400. In some experiments, cis-diol-coated glass 0394 E. coli grown in culture was serially diluted in cold, microbubbles were activated with 0.2M sodium periodate for sterile PBS containing BSA and sodium azide to 5,000 cell/ 90 minutes at room temperature. The product was washed ml. The bacterial suspension was treated with 1% (V/w) anti with water, followed by ethanol and allowed to air-dry at body-coated microbubbles with agitation at room tempera room temperature. This chemistry coated the glass ture for several minutes. The microbubbles were allowed to microbubble surface with amine-reactive aldehydefunctions. float to the surface and 10 ul of underlying liquid layer Subsequent coating with avidin, followed by biotinylated (cleared by floatation of the microbubbles) was removed and antibody and testing, was performed as described above, with plated onto agar media in parallel with untreated, control similar results. samples of bacterial Suspension. Bacterial Suspensions treated with uncoated microbubbles exhibited no reduction in 1-31. (canceled) colonies formed overnight at 37° C. Bacterial suspension 32. A method for generating protein microbubbles for use treated with antibody-coated glass microbubbles exhibit a 50 in affinity isolation or affinity assay comprising: to 100% decrease in colony forming units, which were recov (a) heating a solution of protein; and ered by resuspension and plating. This result confirmed the (b) Sonicating the solution to introduce gas into the solu capture of bacterial cells on glass microbubbles coated with a tion, thereby generating protein microbubbles for use in specific antibody. affinity isolation or affinity assay. 33. The method of claim 32, wherein the gas is air and the Example 11 protein is albumin. 34. The method of claim 32, further comprising stabilizing Coating Epoxy-Glass Microbubbles with Antibody the protein microbubbles with Crsup.+++. 0395 3MTM ScotchLiteTM Glass Bubbles H20/1000 were 35. A method for generating affinity-modified protein washed and suspended in 0.1 M sodium borate, 0.15 M microbubbles for use in affinity isolation or affinity assay sodium chloride, pH 9.0. Avidin was added at 5 mg/ml and the comprising: (a) providing a protein, wherein the protein is suspension was incubated at 4°C. for 48 hours. The avidin coupled to an affinity molecule; (b) heating a solution of coated glass microbubbles were washed in BSA/PBS with protein; and (c) Sonicating the solution to introduce gas into 0.05% sodium azide to remove unbound avidin and stored in the solution, thereby generating affinity-modified protein the same solution. Theavidin microbubbles were coated with microbubbles. biotin-labeled antibody to E. coil 0157:H7 and used to 36. A method for generating microbubbles for use in affin remove bacterial cells from suspension as described above. ity isolation or affinity assay comprising: providing microbubbles; andcoating the microbubbles with an affinity Example 12 molecule. 37. The method of claim 36, wherein the microbubbles are Preparation of Cis-Diol Coated Glass Microbubbles protein microbubbles. 0396 Glass microbubbles with active hydroxyl residues 38. The method of claim 36, wherein the microbubbles are were prepared as described above (see Example 6, above). glass microbubbles. The dried glass microbubbles were silanized by treatment 39. The method of claim 36, wherein the affinity molecule with dry acetone containing 6% 3-glycidoxypropyltri is selected from the group consisting of a receptor, a ligand, a methoxysilane (v/v) for 3 hours at room temperature. Excess nucleic acid and an antibody. silane reagent was removed by washing with acetone, fol 40. The method of claim 36, wherein the affinity molecule lowed by suspension in 0.05 M HCl for 2 hours at 60° C. to is biotin. convert the epoxy function to cis-diol functions. Acid was 41. The method of claim 36, wherein the affinity molecule removed by washing in acetone and the microbubbles were is avidin or streptavidin. US 2011/023.0644 A1 Sep. 22, 2011 13

42. The method of claim 36, wherein the coating comprises 44. The method of claim 36, wherein the coating comprises covalently coupling the affinity molecule to an amine group coupling the affinity molecule to the microbubble through a on the microbubble. heterobifunctional reagent. 43. The method of claim 36, wherein the coating comprises 45-58. (canceled) covalently coupling the affinity molecule to an epoxy group on the microbubble. ck