WO2015200210A1

WO2015200210A1 - Apparatus for use in a magnetic system for the rapid detection or separation of targets of interest in liquid samples

Info

Publication number: WO2015200210A1
Application number: PCT/US2015/036972
Authority: WO
Inventors: Rick SINK; Reha O. Azizoglu; John Groelke; Duane OLSEN; Robert L. Cheek; Adrien Malick; Jasper N. POLLARD
Original assignee: Advanced Animal Diagnostics, Inc.
Priority date: 2014-06-23
Filing date: 2015-06-22
Publication date: 2015-12-30

Abstract

An apparatus (or "container fixture") useful in preparing a liquid sample such as a biological fluid for further analysis, the liquid sample contained in a sample container, is described. The apparatus has: (a) a body having a top portion and a front side wall portion, (b) at least one receptacle formed in the body and extending downward from the body top portion, the receptacle configured to receive the sample container, and with the receptacle having a back side wall portion; (c) at least one magnet connected to the receptacle back side wall portion; and (d) optionally, a retention member connected to the body and configured to force the container against the at least one magnet when the container is positioned in the receptacle.

Description

APPARATUS FOR USE IN A MAGNETIC SYSTEM FOR THE RAPID DETECTION OR SEPARATION OF TARGETS OF INTEREST

IN LIQUID SAMPLES

Rick Sink, Reha O. Azizoglu, John Groelke, Duane Olsen, Robert L. Cheek, Adrien Malick, and Jasper N. Pollard

Related Applications

This application claims the benefit of United States provisional patent application Serial No. 62/015,783, filed June 23. 2014, the disclosure of which is incorporated by reference herein in its entirety.

Field of the Invention

The present invention concerns methods and apparatus useful as an aid for detecting or separating targets of interest such as analytes, including pathogens such as Mycoplasma species, in liquid samples such as biological fluids.

Background of the Invention

The Mycoplasma are a wide-spread group of bacteria. Species such as M pneumoniae and M. genitalium cause disease in humans. Related species cause disease in plants. M. bovis is considered one of the more pathogenic species and causes pneumonia, mastitis, and arthritis in cattle. In research laboratories, Mycoplasma species are frequent contaminants in cell cultures.

Mycoplasma are characterized by the absence of a cell wall. Unfortunately, the most important group of antibiotics, the beta-lactams (which include both the penicillins and the cephalosporins) function by inhibiting cell wall synthesis. With important antibiotics such as these unavailable for the treatment of Mycoplasma infections, there is a need for new and rapid methods and apparatus for the detection of these species so that they may be quickly detected on occurrence and controlled or eradicated before the spread thereof.

Summary of the Invention

Disclosed herein is an apparatus (or "container fixture") useful in preparing a liquid sample contained within a sample container for further analysis or processing. The sample generally has magnetically attractable particles added thereto (for example, particles that have coupled thereto an antibody that binds an analyte in the sample). The apparatus— sometimes also referred to as a "container fixture^"' herein— generally comprises: (a) a body having a top portion and a front side wall portion, (b) at least one receptacle formed in the body and extending downward from the body top portion, the receptacle configured to receive the sample container, and with the receptacle having a back side wall portion; and (c) at least one magnet connected to the receptacle back side wall portion (e.g., for attracting the magnetically attractable particles in the biological sample so that wash steps or the like may be carried out without excessive loss of the particles). The apparatus may optionally further comprise (d) a retention member connected to the body and configured to force the container against the at least one magnet when the container is positioned in the receptacle.

The present invention is explained in greater detail in the drawings herein and the specification set forth below. The disclosures of all United States Patent references cited herein are to be incorporated by reference herein in their entirety.

Brief Description of the Drawings

Figure 1 is a perspective view of an apparatus of the invention, with one sample container in place in its corresponding receptacle, and another sample container exploded therefrom.

Figure 2 is a perspective view of the apparatus of Figure 1, with both sample containers removed, and one magnet shown exploded from one of the two receptacles.

Figure 3 is a perspective view of the apparatus of Figures 1-2, with both sample containers in place.

Detailed Description of Illustrative Embodiments

The present invention now will be described more fully hereinafter with reference to the accompanying figures, in which embodiments of the invention are shown. This invention may, however, be embodied in many alternate forms and should not be construed as limited to the embodiments set forth herein. Like numbers refer to like elements throughout the description of the figures.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein. the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. As used herein the term "and/or" includes any and all combinations of one or more of the associated listed items.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

1. Definitions.

"Subject" as used herein includes both human and animal subjects for veterinary purposes, as well as plants for agricultural purposes. Examples of animal subjects include, but are not limited to, mammalian subjects such as dog, cat, cow, sheep, goat, horse, and pig subjects, fish such as salmon, trout, and tilapia, and avian subjects such as chicken, turkey, duck, geese, quail, and pheasant.

"Liquid sample^" as used herein may be any liquid suspected of containing one or more analytes. The liquid sample is typically an aqueous sample, and may be provided as a single phase or multi-phase sample (e.g.. an emulsion, dispersion, or suspension of solid or liquid particles in a (typically aqueous) continuous phase). For example: plant or animal tissue, fecal material, or a solid food sample, may be homogenized in an aqueous solution to provide a liquid sample; a solid sample such as a soil sample may be rinsed in an aqueous rinse or wash solution such as water or buffer solution, and the rinse or wash solution used as the aqueous sample. A water sample may be taken from a pond (e.g., an aquaculture farm for raising fish, crustaceans or the like), ocean, stream, river or the like, optionally diluted, and used as the liquid sample. In some embodiments, the liquid sample is a biological fluid. In some embodiments the liquid sample is a growth media such as cell or tissue culture media. In some embodiments the liquid sample is undiluted; in other embodiments the liquid sample is diluted with the addition of an additional liquid such as a buffer.

"Biological fluid" as used herein refers to a liquid solution or suspension comprising material collected from or excreted by a subject. Examples include, but are not limited to, milk, colostrum, secretions, whole blood, blood plasma, urine, mucus, lymph, throat and nasal swabs, vaginal swabs, sputum, bronchial lavage fluid, etc.. from human and animal subjects; sap, nectar or juice from plants, tissue homogenates of any thereof, and fractions of any thereof such as blood plasma. The fluid may be taken from a vector such as an insect that carries the pathogen, or may comprise a tissue homogenate of such vector. In some embodiments the biological fluid may be undiluted while in other embodiments the biological fluid may further comprise or contain one or more diluents or additives such as washes, rinses, and/or other diluents (e.g., aqueous diluents such as saline solutions) in any suitable volume ratio of diluents to biological fluid (e.g., from 4: 1 , 3: 1 , 2: 1, or 1 :1 to 1 :2, 1 :2, 1 :3, 1 :4, etc.), along with other additives such as anticoagulants, preservative, salts, buffers, detergents, surfactants, etc. The biological fluid is optionally but preferably complete or whole (e.g., whole milk or whole colostrum), which has not been subjected to separation steps such as filtering, fractioning, centrifuging, chromatography, etc.

"Milk" as used herein generally refers to mammalian milk of any species (e.g.. cow. goat, human, etc.). The milk may be raw or pasteurized, depending upon the particular purpose of the test. Milk may be whole milk, low-fat or reduced fat milk, or skim milk. Milk may optionally be diluted (typically with an aqueous diluent such as distilled water, saline solution, or buffer solution), as discussed above.

"Colostrum" as used herein is a form of milk produced by mammals in the first few days after birth, that may be higher in antibodies (for imparting passive immunity to offspring). The term "colostrum" as used herein includes "secretions" as described below.

"Secretions" (or "mammary gland secretions" ) as used herein is a form of milk produced by mammals just prior to giving birth. Such secretions are sometimes also referred to as "colostrum" but in the present application "secretions^" refers to the type of milk produced prior to the subject giving birth, while colostrum refers to the type of milk produced just after the subject giving birth.

"Target entity^" as used herein refers to compounds, organisms, and viruses of interest. In some embodiments the target is referred to as an analyte, although in other embodiments the target may be one for which separation is desired for reasons other than subsequent analysis of that specific target.

"Analyte" (also referred to as "measurands^") as used herein includes any suitable target of analysis or target of measurement. Such analytes, measurands, or targets as used herein may be any suitable compound or cell to which an antibody will bind, including but not limited to proteins, peptides, nucleic acids, toxins, and pathogens.

"Toxin^" as used herein includes, but is not limited to, mycotoxins and bacterial toxins (e.g., exotoxins, enterotoxins, and/or endotoxins).

" ycotoxin^" as used herein includes, but is not limited to, aflatoxins (e.g., aflatoxin Bl . B2, Gl, and G2), vomitoxin, ochratoxins (e.g., ochratoxin A. B, and C), citrinin, ergot alkaloids, and fusarium toxins (e.g., fumonisins, and trichothecencs).

"Enterotoxin^" as used herein includes, but is not limited to. Staphylococcus aureus enterotoxin and Escherichia coli enterotoxin.

"Pathogen" as used herein may be any pathogen, including viral, fungal (including yeast), bacterial (including Gram negative and Gram positive bacteria), and protozoan pathogens. In some embodiments, the pathogen is a mollicute such as a mycoplasma.

"Mollicute^" as used herein refers to a class of bacteria characterized by the absence of a cell wall. Orders within the class Molicutes include Acholeplasmatales, Anaeroplasmatales, Entomoplasmatales, Haloplasmatales, and Mycoplasmatales. Examples include, but are not limited to Mycoplasma, Ureaplasma, Acholeplasma, Spiroplasma, and Phytoplasma. "Slow growing pathogen," as used herein, refers to microbial pathogens that require more than 10, 24 or in some embodiments 48 hours to double in population when grown in culture (as compared to, for example, bacteria such as E. coli, which can double in population in 2 to 3 hours). Examples of slow growing pathogens include, but are not limited to, Borrelia, Pediococcus, Mycoplasma, and Mycobacteria, See, e.g., PCT Application No. WO2002074991.

"Mycoplasma " as used herein refers to a genera of bacteria within the order Mycoplasmatales mat lack a cell wall. Examples include, but are not limited to, mycoplasma bovis, mycoplasma genitalium, mycoplasma hominis, mycoplasma hyopneumoniae, mycoplasma laboratorium, mycoplasma ovipneumoniae, mycoplasma pneumonia, mycoplasma haemofelis, mycoplasma californicum, mycoplasma bovigenitalium, etc.

"Ureaplasma" as used herein also rfers to a genera of bacteria within the order Mycoplasmatales that are urease positive. Examples include, but are not limited to, ureaplasma parvum and ureaplasma urealyticium.

"Mycobacteria" as used herein includes, but is not limited to, Mycobacterium simiae, Mycobacterium bovis, Mycobacterium szulgai, Mycobacterium malmoense, Mycobacterium intracellular e, Mycobacterium avium, Mycobacterium gordonae, Mycobacterium africanum, Mycobacterium tuberculosis, Mycobacterium gastri, Mycobacterium marinum, Mycobacterium microti, Mycobacterium asiaticum, Mycobacterium scrofulaceum, Mycobacterium branderi, Mycobacterium paratuberculosis , and Mycobacterium kansasii. See, e.g., European Patent Application No. EP1098003.

"Borrelia" as used herein includes, but is not limited to, B. burgdorferi, B. afzelii, and B. garinii (the major species causing Lyme disease), along with other species such as B. recurrentis, B. hermsii, B. parkeri, B. miyamotoi, etc. , which may cause borreliosis or relapsing fever borreliosis.

"Binding moiety" as used herein refers to any suitable compound that binds (e.g., selectively and/or specifically binds) the target, including but not limited to nucleic acids, proteins, peptides, carbohydrates, etc. In some embodiments the binding moiety is an antibody.

"Antibodies" as used herein refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, IgE, and IgY, and including combinations thereof. The antibodies may be monoclonal or polyclonal and may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, avian (e.g., chicken) or reptile), or may be chimeric antibodies. See, e.g., W. Bergter et al., US Patent Application Publication No. 2004/0236076; M. Walker et al., Molec. Immunol. 26, 403-11 (1989). The antibodies may be recombinant monoclonal antibodies produced according to the methods disclosed in Reading U.S. Patent No. 4,474,893, or Cabilly et al.. U.S. Patent No. 4.816.567. Antibody fragments included within the scope of the present invention include, for example. Fab, F(ab')2. and Fc fragments, and the corresponding fragments obtained from antibodies other than IgG. Such fragments can be produced by known techniques. Thus included are both truncated (yolk) IgY and complete or intact (plasma) IgY.

"Fluorescent stain" as used herein may be any suitable stain for fluorescence or epitluorscence microscopy, including but not limited to acridine orange, Astrazon Orange G (source: Sigma Aldrich), SYBR Green I (source: Life Technologies), SYTOX Green (source: Life Technologies), etc. Additional examples include but are not limited to those set forth in US Patents Nos. 8,263,392; 8,168,386; 8,159,676; 8,084,260; 7,638,290 and 7,236,236. In some embodiments the fluorescent stain is a fluorescent intercalating stain. The fluorescent stain may be utilized per se or may be contained within a carrier such as a particle to enhance the delivery thereof, and/or may be optionally coupled to a binding ligand for the particular organism, virus or particle being stained to enhance the selectivity thereof. See, e.g., M. Bally et al., A virus biosensor with single virus-particle sensitivity based on fluorescent vesicle labels and equilibrium fluctuation analysis, Biointerphases 8:4 (2013).

"Magnetically attractable particles" as used herein may be regular or irregular in shape. While particles of any suitable diameter or average diameter may be used, in some embodiments the particles are at least 0.1 , 0.5, or 1 micron in diameter, up to 3, 5. 10, or 20 microns in diameter, or more. The particles may contain diamagnctic, ferromagnetic, ferrimagnetic, paramagnetic or superparamagnetic material. Superparamagnetic material responds to a magnetic field with an induced magnetic field without a resulting permanent magnetization. Magnetic particles based on iron oxide are for example commercially available as DYNABEADS® from Dynal Biotech, as magnetic MicroBeads from Miltenyi Biotec, as magnetic porous glass beads from CPG Inc., as well as from various other sources, such as Roche Applied Science, BIOCLON, BioSource International Inc., micromod. AMBION, Merck, Bangs Laboratories, Polysciences. or Novagen Inc.. to name only a few. Magnetic nanoparticles based on superparamagnetic Co and FcCo, as well as ferromagnetic Co nanocrystals have been described, for example by Hutten, A. et al. (J. Biotech. (2004), 1 12, 47-63). See, e.g., US Patent No. 8,216,855; see also US Patent No. 8,318,093. The magnetically attractable particles preferably have a binding moiety such as an antibody bound thereto to bind the target of interest.

"Magnet" as used herein may be any suitable magnet, including electromagnets and permanent magnets {e.g., magnets formed from ferromagnetic materials like annealed iron, alnico, ferrite, flexible and ceramic magnets, etc., including composites of a magnet particles and a non-magnetic material such as a polymer)). In some embodiments the magnet is preferably a rare-earth magnet such as a samarium-cobalt magnet or a neodymium magnet (also called neodymium-iron- boron or "NIB" magnets). In embodiments that contain more than one magnet, the magnets may be the same or different.

2. Antibodies and analytes for detection.

As noted above, the present invention may be utilized for detecting any of a variety of analytes to which antibodies may be raised, and to which antibodies bind. In some embodiments, the analyte is, or the analytes are, pathogens or toxins.

Numerous pathogens are known. See, e.g., US Patent No. 7,945,393. Examples of pathogens (e.g., human pathogens or those of animals or plants) that can be assessed using the methods described herein include bacteria (including eubacteria and archaebacteria), eukaryotic microorganisms (e.g., protozoa, fungi, yeasts, and molds) viruses, and biological toxins (e.g., bacterial or fungal toxins or plant lectins). Specific examples of such pathogens include protozoa of the genus Cryptosporidium, protozoa of the genus Giardia. bacteria of genera such as Escherichia, Escherichia coli. Escherichia coli 157, Yersinia, Francisella, Brucella, Clostridium, Burkholderia. Chlamydia, Coxiella. Rickettsia, Vibrio, Leptospira, Enterococcus, Staphylococcus, Streptococcus, methicillin-resistant staphylococcus (MRSA), Enterobacter, Corynebacterium, Pseudomonas, Acinetobacter, Klebsiella, and Serratia. Assessable organisms include at least Escherichia coli. Yersinia pest is. Francisella tularensis, Clostridium perfringens, Burkholderia mallei. Burkholderia pseudomallei. cryptosporidia microorganisms, Tularemia (Francisella tularensis), Brucellosis (Brucella species). Chlamydia psittaci (psittacosis), Coxiella burneti (Q fever), Rickettsia prowazeki (Typhus fever). Vibrio vulnificus. Vibrio enteral yticus. Vibrio fischii. Vibrio cholera, Enterococcus faecal is. Staphylococcus epidermidis, Staphylococcus aureus, Enterobacter aerogenes, Corynebacterium diphtheriae, Pseudomonas aeruginosa, Acinetobacter calcoacetieus, Klebsiella pneumoniae. Serratia marceseens, Candida albicans, Microsporum audouini. Microsporum canis, Microsporum gypseum. Trichophyton mentagrophytes var. mentagrophytes. Trichophyton mentagrophytes var. interdigitale. Trichophyton rubrum. Trichophyton tonsurans. Trichophyton vcrrucosum, and Epidermophytum floccosum. Streptococcus (including Strep A, B, C, G) filoviruses such as Ebola and Marburg viruses, navi ruses such as Lassa fever and Machupo viruses, alphaviruses such as Venezuelan equine encephalitis, eastern equine encephalitis, and western equine encephalitis, rotaviruses, calciviruses such as Norwalk virus, and hepatitis (A, B. and C) viruses.

Additional examples of pathogens that can be detected by the methods and apparatus of the present invention include, but are not limited to. Bacillus anthracis. Bartonella quintana. Brucella melitcnsis, Burkholderia mallei. Burkholderia pseudomallei. Chlamydia psittaci. Clostridium botulinum, Clostridium perfringens. Coxiella burnetti, enterohemorrhagic Escherichia coli, Francisella tularensis. Rickettsia mooseri. Rickettsia prowasecki, Rickettsia rickettsii, Rickettsia tsutsugamushii. Salmonella typhi. Salmonella. Shigella. Shigella dysenteriae. Vibrio cholerae. Yersinia pestis, Coccidioides immitis, Histoplasma capsulatum, chikungunya virus, Congo-Crimean haemorrhagic fever virus, dengue fever virus. Eastern equine encephalitis virus, ebola virus, equine morbillivirus. hantaan virus, Japanese encephalitis virus, junin virus, lassa fever virus, Epstein Barr virus (infectious mononucleosis), lymphocytic choriomeningitis virus, machupo virus, marburg virus, monkey pox virus, Murray valley encephalitis virus, nipah virus. Omsk hemorrhagic fever virus, oropouche virus. Rift valley fever virus, Russian Spring-Summer encephalitis virus, smallpox virus. South American hemorrhagic fever viruses, St. Louis encephalitis virus, tick-borne encephalitis virus. Variola virus. Venezuelan equine encephalitis virus. Western equine encephalitis virus, white pox virus, yellow fever virus, botulinum toxins. Clostridium perfringens toxins, microcystins (Cyanginosins). Shiga toxin, verotoxin, Staphylococcal enterotoxin B, anatoxin A, conotoxins, palytoxin, saxitoxin, tetrodotoxin, stachybotrys toxins, anatoxins, trichothecenes, satratoxin H, T-2 toxin, and ricin. Other examples include Abrus precatorius lectin. African swine fever virus, avian influenza virus, banana bunchy top virus, bluetongue virus, camelpox virus, cholera toxin, Clostridium perfringens, Clostridium tetani, Cryptosporidium parvum, Deuterophoma tracheiphila, Entamoeba histolytica, ergot alkaloids, Escherichia coli 0157, foot and mouth disease virus, Giardia, Giardia lamblia, goat pox virus, hendra virus, hepatitis A virus, hog cholera virus (also known as classical swine fever virus), human immunodeficiency virus, infectious conjunctivitis virus, influenza virus (including influenza A, influenza B, and influenza C viruses), Kyasanur Forest virus, Legionella pneumophila, louping ill virus, lyssaviruses, Adenia digitata lectin (modeccin), Monilia rorei, Naegleria fowleri, nipah virus, Murray Valley encephalitis virus, Mycoplasma mycoides, newcastle disease virus, oropouche virus, peste des pelits ruminants virus, porcine enterovirus 9, powassan virus, pseudorabies virus, rinderpest virus, rocio virus, group B rotaviruses. Salmonella paratyphi, sheeppox virus. St. Louis encephalitis virus, substance P, Serratia marcescens, Tcschen-Talfan virus, tetanus toxin, vesicular stomatitis virus, Visctim album lectin 1 (Viscumin), Adcna volkensii lectin (volkensin). West Nile virus. Xanthomonas campestris oryzae, Xylella fastidiosa, and Yersinia pseudotuberculosis, and porcine epidemic diarrhea virus.

Examples of plant pathogens that can be assessed by the methods and apparatus of the present invention include, but are not limited to, Burkholderia solanacearum, citrus greening disease bacteria, Erwinia amylovora, Xanthomonas albilineans, Xanthomonas axonopodis pv. citri, Bipolaris (Helminthosporium) maydis. Claviceps purpurea, Colletotrichum coffcanum virulans. Cochliobolus miyabcanus. Dothi stroma pini. Fusarium oxysporum. Microcystis ulei, Neovossia indica, Peronospora hyoscyami, Puccinia erianthi, Puccinia graminis, Puccinia graminis f. sp. tritici, Puccinia striifonnis. Pyricularia grisea, Sclerotinia scierotiorum, Sclerotium rolfsii, Tilletia indica, Ustilago maydis, Phytophthora infestans, and Fiji disease virus.

In some embodiments, the pathogen is detected directly. In other embodiments, the pathogen is indirectly detected by detecting the presence of a toxin which the pathogen produces, whether or not the pathogen itself remains present. Polyclonal antibodies used to carry out the present invention may be produced by any suitable technique, such as by immunizing a suitable animal (e.g., rabbit, goat, etc.) with an antigen (e.g., the analyte, optionally coupled to an adjuvant), collecting immune serum from the animal, and separating the polyclonal antibodies from the immune serum, in accordance with known procedures. In some embodiments, antibodies can be collected from eggs, in accordance with known techniques.

Monoclonal antibodies used to carry out the present invention may be produced in a hybridoma cell line according to the technique of Kohler and Milstein, Nature 265, 495-97 (1975). For example, a solution containing the appropriate antigen may be injected into a mouse and. after a sufficient time, the mouse sacrificed and spleen cells obtained. The spleen cells are then immortalized by fusing them with myeloma cells or with lymphoma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells. The hybridoma cells are then grown in a suitable media and the supernatant screened for monoclonal antibodies having the desired specificity. Monoclonal Fab fragments may be produced in Escherichia coli by recombinant techniques known to those skilled in the art. See, e.g., W. Huse, Science 246, 1275-81 (1989). Antibodies specific to the analyte may also be obtained by phage display techniques known in the art.

Once produced, the antibody is immobilized on the magnetically attractable particles in accordance with known techniques or variations thereof that will be apparent to those skilled in the art. See, e.g., US Patent Nos. 8,101,155; 8,043,821; 8,003,766; 7,829,294; 7,695,609; 7,288,253; and 7,247,453.

3. Apparatus.

A non-limiting example of the invention is shown in Figures 1-3 herein. As shown, the invention provides an apparatus (or "container fixture") 20 useful in preparing a liquid sample for further analysis, the biological sample contained in a sample container 10. The sample generally has magnetically attractable particles added thereto, which particles have coupled thereto an antibody that binds an analyte in the sample. The apparatus comprises: (a) a body having a top portion 21 and a front side wall portion 22, (b) at least one receptacle 23 formed in the body and extending downward from the body top portion, the receptacle configured to receive the sample container, and with the receptacle having a back side wall portion 25; (c) at least one magnet 30 connected to the receptacle back side wall portion 25; and (d) optionally, a retention member 31 connected to the body and configured to force the container against the at least one magnet when the container is positioned in the receptacle. Alternatively, the receptacle may be so dimensioned as to force the container against the at least one magnet, particularly when the container is formed of a resilient or flexible polymer material.

In the illustrated embodiment, the receptacle is configured so that the sample container 10 (which may be cylindrical in a convenient preferred embodiment utilizing commonly available sample containers) is rotatable when partially inserted therein. In this case, the cylindrical container may have a projecting member 11 formed thereon (a living hinge connecting the container cap 12 to the container body 13 in the illustrated embodiment), the apparatus may further comprise at least one alignment member 24 connected to the body, operatively associated with the at least one receptacle, and configured to receive the projecting member and restrain the sample container from rotating in the receptacle. In the illustrated embodiment the alignment member is a rectangular notch that matches the shape of the living hinge, but any suitable shape can be employed as long as the features on the container mate with the features on the fixture.

As discussed further below, the at least one receptacle 23 may comprise a pair of parallel receptacles (with the addition of still additional receptacles, e.g., totaling three or four receptacles in a fixture, being optional).

The retention member is a coil spring 31 in the illustrated embodiment, but may be any suitable retention member, such as an elastic band, a screw clamp, or combination thereof. Separate retention members may be provided for each receptacle in the fixture, or (as illustrated) the retention member may be a single retention member configured in cooperation with both of the pair (or more) of receptacles.

While any of a variety of magnet shapes and orientations may be used, preferably the magnet is rectangular and hence has a length dimension. The receptacle likewise has a length axis, and in one preferred embodiment the magnet length dimension is substantially parallel to the receptacle length axis (and hence to the length axis of the container when the container is in the receptacle).

The magnets 30 preferably have flat front and back surfaces, which are oriented in the fixture receptacle substantially tangential to the radius of the (generally cylindrical) receptacle, his serves to enhance the contact of the magnet to the receptacle side wall.

Preferably, the magnets are rare earth magnets. The magnets may be secured into the fixture body (which is typically a non-magnetic polymer materials such as polycarbonate or acrylonitrile butadiene styrene (ABS) by any suitable means, such as by the provision of one or more "keeper" magnets or ferromagnetic elements behind the magnet, adhesive, or combinations thereof.

In a preferred embodiment, the at least one magnet 30 in each receptacle 23 comprises a pair of magnets (optionally but preferably identical to one another in shape and composition) connected to each of the at least one receptacle back side wall portion, with the pair of magnets parallel to and spaced apart from one another in each of the at least one receptacles {e.g., so that the retention member forces the sample container substantially equally against both of the pair of magnets ).

While in some embodiments a single magnet in each receptacle may be used, and indeed such was our first embodiment of the fixture, we have found that a pair of magnets provides the best contact with the container side wall, and appear to make wash steps more effective since the two magnets separate the magnetically attractable particles in the sample into two distinct lines, each about half the linear density of a line that would be produced by a single magnet. Consequently, it appears the particle lines are more exposed to the wash solution and can be washed more completely and'or more rapidly than when a single magnet is employed.

In a preferred embodiment, the North pole of the at least one magnet (or both magnets) in one of the pair of receptacles faces in the opposite direction of the North pole of the at least one magnet (or both magnets) in the other of the pair of receptacles (that is, in the illustrated embodiment, both magnets in one receptacle have their North poles facing forward, and both magnets in the other receptacle have their South poles facing forward). Since a repetitive analytic or diagnostic procedure may employ numerous fixtures, this allows a plurality (e.g., 2. 4, 6. or 8 up to 20, 30. 40 or more) of the apparatus to be magnetically coupled to one another (e.g., in front-to-back and/or sidc-to-side arrangement or orientation), thus simplifying and facilitating handling of the fixtures by the user or operator. For this purpose it will be appreciated that the members of the plurality should be in the same general shape and configuration that allows them to stably abut one another when magnetically attracted. This can be achieved by providing substantially flat front, side, or back surface portions, or appropriate "bumper^" portions on such surfaces, or by providing cooperating or mating features on certain portions of the fixture body (e.g., a convex left body side portion and a corresponding concave right body side portion, or vice versa; corresponding projecting elements, etc.).

In addition, in the embodiment employing a pair of magnets in each receptacle, the poles (North and South) of both members of the pair preferably face in the same direction in each receptacle. In addition to facilitating the coupling and handling of fixtures as noted above, this appears to aid in aligning the magnetically attractable particles into separate groups or lines, thus facilitating washing as noted above.

4. Methods of use.

Also provided herein is a method useful as an aid to detecting or separating a target of interest (e.g., an analyte) in a liquid sample, comprising the steps of:

(a) combining in a sample container a liquid sample suspected of containing the analyte and magnetically attractable particles, the magnetically attractable particles having anti-analyte antibodies coupled thereto, under conditions in which the analyte, when present, binds to the magnetically attractable particles;

(b) positioning at least one first magnet adjacent the sample container so that the magnetically attractable particles are immobilized against an internal wall of the sample container;

(c) decanting the sample from the sample container, the magnetically attractable particles remaining immobilized against the internal wall;

(d) optionally washing the magnetically attractable particles in the sample container; and then

(e) separating the external magnet from the sample container so that magnetically attractable particles are released into the interior of the sample container, (e.g.. from where they may then be decanted for further analysis).

In some embodiments of the method, including but not limited to those carried out with the aid of the fixture or apparatus described herein, the positioning step (b) is carried out by clamping the first magnet to an external wall (e.g.. a side or bottom wall) of the sample container. In some embodiments, the washing step (d) is carried out by (i) adding a wash solution to the sample container, (ii ) washing the magnetically attractable particles therein, (iii) decanting the wash solution from the vessel with the magnetically attractable particles immobilized against a side wall of the sample container, and (iv) optionally repeating steps (i) through (iii), as many times as desired or necessary for the particular assay system employed. While the magnet (or magnetss) may be separated during the wash steps (of course being returned prior to decanting to avoid excessive loss of the particles), the more rapid procedure is to leave the magnet in place, and indeed to retain alignment of the container with the magnet(s) throughout the wash steps.

As noted above, when the at least one first magnet is rectangular, the magnetically attractable particles are immobilized on the internal wall o the sample container in the form of a line, providing the user with visual confirmation of their presence. When the at least one magnet comprises a pair of magnets spaced apart from one another, the magnetically attractable particles are likewise magnetically immobilized in a pair of lines spaced apart from one another on the internal wall of the sample container.

When desired, agitation of the fixture, to agitate the biological sample(s). may be achieved by any suitable manual or automated technique that imparts motion of the magnetically attractable particles relative to the biological fluid, or vice versa. In contrast to the centrifugation used to concentrate and separate the carrier as described in US Patent No. 5,776,710 to Levinc ct al., agitation in the present invention is carried out in a manner which mixes or disperses the sample, and more particularly or mixes or disperses the analyte throughout the liquid sample. Agitation is typically carried out at ambient or room temperature, and may be carried out for any suitable time. In some embodiments, agitation is carried out for a time of 10, 20 or 30 minutes, up to 1 , 2 or 3 hours, or more.

Those skilled in the art will be familiar with numerous specific quantitative and qualitative detection and assay formats and variations thereof which may be useful for carrying out the method disclosed herein. See generally E. Maggio, Enzyme-Immunoassay, (1980)(CRC Press, Inc.. Boca Raton. FL); see also U.S. Patent No. 4,727,022 to Skold et al. titled "Methods for Modulating Ligand-Receptor Interactions and their Application." U.S. Patent No. 4,659.678 to Forrest et al. titled "Immunoassay of Antigens," U.S. Patent No. 4.376,1 10 to David ct al., titled "Immunometric Assays Using Monoclonal Antibodies." U.S. Patent No. 4,275.149 to Litman et al., titled "Macromolecular Environment Control in Specific Receptor Assays," U.S. Patent No. 4,233,402 to Maggio et al., titled "Reagents and Method Employing Channeling," and U.S. Patent No. 4,230,767 to Boguslaski et al., titled "Heterogenous Specific Binding Assay Employing a Coenzyme as Label." Applicants specifically intend that the disclosures of all U.S. Patent references cited herein be incorporated herein by reference in their entirety.

When the sample comprises cells, viruses or the like to be imaged and/or counted, the cells may be stained by a suitable stain, including fluorescent stains such as acridine orange (see, e.g., US Patent No. 3,883,247).

In some embodiments, the methods and device achieve detectable capture of analytes such as Mycoplasma bovis or Staphylococcus aureus present in a biological fluid such as milk at a concentration of as little as 10 pathogens per milliliter of biological fluid after one hour of agitation.

The present invention is explained in greater detail in the following non- limiting Examples.

EXAMPLE 1

Antibody Preparation

Anti- Mycoplasma bovis antibody IgY is conjugated with biotin by methods set forth below:

1. Dialyze the antibody in modification buffer. Prepare stock solution of Biotin in DMSO by dissolving 1 mg Biotin in 150 μΐ DM SO.

2. Add 5 molar excess of Biotin stock solution to the dialyzed antibody solution.

3. Incubate the antibody-Biotin mixture at room temperature.

4. Dialyze the antibody-Biotin solution in IX dialysis buffer.

5. Take UV spectrophotometric readings at OD280 & OD354 to determine Biotin / antibody molar ratio and final protein concentration.

EXAMPLE 2

Magnetic Particle Testing Protocol

1. Transfer 400 μΐ magnetic particles into a 1.5 ml micro centrifuge tube. 2. Place tube in magnetic rack for three minutes until particles form a side pellet. Remove and discard supernatant by pi pet.

3. Wash the particles 2 times with 1 ml of binding/wash buffer, using the magnetic rack as in step #2 above along with mixing of particles by vortexing of tube during wash step. Form pellet with magnet rack, remove and discard second wash before proceeding to step #4.

4. Add 300 μΐ 2X binding/wash buffer to tube; resuspend particles .

5. Add 298 μΐ of biotinylated anti-Mycoplasma bovis IgY antibody to the particles (the antibody is +/1 biotinylated and could be IgG or IgY).

6. Mix particle/antibody solution on a gyrating mixer at room temperature for 1 hour.

7. Place tube in magnetic rack for three minutes until particles form a side pellet.

Remove supernatant by pipet and retain.

8. Wash the particles 3 times with 1 ml of binding/wash buffer. Form pellet with magnet rack, remove and discard third wash before proceeding to step #9

9. Add 1 ml particle buffer to tube, and resuspend particles. Particles are now ready to use in Mycoplasma bovis immunoassay..

10. Add 2 ml of reaction buffer and 2 ml of water to a 5 ml microcentrifuge tube.

1 1. To the 5 ml reaction tube, add Mycoplasma bovis organisms from a bacterial slock .

12. Add Acridine Orange stain to the 5 ml tube.

13. Mix the solution in the 5 ml tube on a gyrating mixer.

14. Tape the magnet to the outside top of the cap of the 5 ml tube and gyrate slowly until particles form a pellet.

15. Carefully open the cap of the 5 ml tube and remove the reaction mixture supernatant by pipet and retain.

16. Add 1 ml of wash buffer to the 5 ml tube, close cap. remove magnet to resuspend the particles, and invert the tube several times by hand to wash the particles. .

17. Add the resuspended particles to the 5 ml tube.

18. Tape the magnet to the outside top of the cap of the 5 ml tube and invert the tube several times by hand until particles form a pellet. 19. Carefully open the cap of the 5 ml tube and remove the wash supernatant by pipet and discard.

20. Wash the magnetic particles two more times by repeating steps #17 - #19 twice more: after the third wash, keep the tube open with the pellet in the cap. 21. Add Wash Buffer to the pellet in the cap of the 5 ml tube and resuspend the particles.

22. Dispense the resuspended particle solution onto a microscope slide and cover with a cover slip.

23. Visualize and capture images of the Mycoplasma bovis organisms on the particles using a fluorescent Nikon microscope using the LED on maximum, with various filter set combinations, microscope settings, and magnifications to obtain the best images.

EXAMPLE 3

Sandwich Assay with Sequential

Addition of Capture and Detector Reagents

1. Use antibody-labeled magnetically attractable beads as described in Example 2 above (or any of a variety of other antibodies that bind the analyte to be detected, the antibodies linked to any suitable bead or microparticle by any suitable coupling technique).

2. Place a small volume of antibody-magnetic bead conjugate in a sample tube with a reaction buffer and the biological sample (e.g. milk).

3. Mix the contents of the sample tube (e.g., by shaking, rocking, stirring, agitation etc.) to allow the beads to bind (via their associated antibody) to the target analyte in the sample.

4. Place the tube on an external magnet to concentrate the beads, with their associated target, by magnetic attraction onto the wall of the tube adjacent the magnet. The external magnet may be hand-held, or more preferably mounted within a fixture that holds the tube in place adjacent the external magnet, as described herein.

5. With the tube held adjacent the external magnet, decant the volume of sample and buffer, leaving the magnetically attractable particles behind in the tube, held against the tube wall by the external magnet. Then, add a wash buffer to the tube. mix the contents again, and again decant the wash buffer. Wash steps may be repeated as needed.

6. Optionally, but in some embodiments preferably, separate the tube from the external magnet, thereby releasing the capture beads (with their associated analyte bound thereto) to move freely within the sample tube.

7. Add a wash buffer (which may or may not be the same as the previous reaction buffer) to the sample tube, which second buffer contains a second antibod that binds the analyte, which second antibody is labeled with a suitable detectable group (e.g., a fluorescent or phosphorescent group, coupled to the antibody, or carried by a particle which is coupled to the antibody ).

8. Mix the contents of the tube to further remove the beads from the tube side wall, and allow to incubate for a time sufficient for the second antibody to bind to the analyte.

9. Preferably, t e sample tube is attached to a read surface above a magnet so that the magnetically attractable beads are concentrated from the solution onto the read surface, carrying with them the second antibody and its associated detectable group, with the analyte "sandwiched^"' therebetween.

10. Some or all of the remaining liquid buffer may then be removed, and the presence or quantity of the magnetically attractable bead-analyte-labelled antibody "sandwich" then measured or detected with a suitable reader or microscope.

EXAMPLE 4

Sandwich Assay with Concurent

Addition of Capture and Detector Reagents

1. Use antibody- labeled magnetically attractable beads as described in step 9 of Example 2 above (or any of a variety of other antibodies that bind to the analyte to be detected, linked to any suitable bead or microparticle by any suitable coupling technique). Use second antibody that binds the analyte. which second antibody is labeled with a suitable detectable group (e.g., a fluorescent or phosphorescent group, coupled to the antibody, or carried by a particle which is coupled to the antibody).

2. A small volume ot antibody-labelled magnetically attractable particles is placed in a sample tube, along with buffer solution, biological sample (e.g., milk), and the labelled second antibody. 3. The contents of the tube is mixed as described above to allow both the beads and the second antibody to bind the analyte and form a "sandwich" thereof.

6. Separate the tube from the external magnet, thereby releasing the capture beads (with their associated analyte and labelled antibody bound thereto) to move freely within the sample tube .

7. Add a further wash butTer solution and mi the contents o the tube to further remove the beads from the tube side wall.

8. Preferably, the sample tube is attached to a read surface above a magnet so that the magnetically attractable beads are concentrated from the solution onto the read surface, carrying with them the second antibody and its associated detectable group, and the analyte "sandwiched" therebetween.

9. Some or all of the remaining liquid buffer may then be removed, and the presence or quantity of the magnetically attractable bead-analyte-labelled antibody "sandwich^" is then measured or detected with a suitable reader or microscope.

EXAMPLE 5

Detection of Analyte with a Nucleic Acid Stain

Steps 1 -5 may be carried out in like manner as in Example 3 above, with analytes such as cells and viruses that contain nucleic acids. After the final wash step, the a final buffer is added to the sample tube and the tube separated from the magnet.

6. Mix the contents of the tube to further remove the beads from the tube side wall, and allow to incubate for a time sufficient for the second antibody to be labeled to bind to the analyte. 7. Preferably, the sample tube is attached to a read surface above a magnet so that the magnetically attractable beads are concentrated from the solution onto the read surface, carrying with them the analyte and the labeled second antibody.

8. Some or all of the remaining liquid buffer may then be removed, and a fluorescent nucleic acid stain (SYBR Green I, Acridine Orange, etc. ) is added in a manner in which it binds to any nucleic acids in the analyte and gives a signal different from the labeled antibody.

9. The presence or quantity of the target analyte is then detected or measured on an appropriate reader or microscope by detecting the absence or presence, and if present optionally also the amount, of nucleic acid slain on the read surface.

The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

THAT WHICH IS CLAIMED IS:

1. A method useful as an aid to detecting or separating an target of interest (e.g., an analyte) in a liquid sample, comprising the steps of:

(a) combining in a sample container a liquid sample suspected of containing said target of interest, and magnetically attractable particles, under conditions in which said target of interest, when present, binds to said magnetically attractable particles;

(b) positioning at least one first magnet adjacent said sample container so that said magnetically attractable particles are immobilized against an internal wall of said sample container;

(c) decanting said sample from the sample container, the magnetically attractable particles remaining immobilized against said internal wall;

(d) optionally washing said magnetically attractable particles in said sample container; and then

(e) separating said external magnet from said sample container so that said magnetically attractable particles are released into the interior of the sample container with said target of interest bound thereto (e.g., from where they may then be decanted for further analysis).

2. The method of claim 1 , wherein said positioning step (b) is carried out by clamping the first magnet to an external wall (e.g., a side or bottom wall ) of the sample container.

3. The method of claim 1 or 2. wherein said washing step (d) is carried out by (i) adding a wash solution to said sample container, (ii) washing said magnetically attractable particles therein, (iii) decanting said wash solution from said vessel with the magnetically attractable particles immobilized against a side wall of said sample container, and (iv) optionally repeating steps (i) through (iii).

4. The method of claim 1 to 3, wherein said analyte comprises a Mycoplasma or a Ureaplasma.

5. The method of claim 4, wherein said analyte comprises M. pneumoniae, M. genitalium, M. bovis, M. californicum, M. bovigenitalium, U. parvum and U. urealyticium.

6. The method of claim 1 to 5. wherein said magnetically attractable particles have an antibody bound thereto, which antibody specifically binds said target of interest.

7. The method of claim 6. wherein said liquid sample comprises a biological fluid (e.g., milk, colostrum, whole blood, blood plasma, urine, nasal swab fluid, vaginal swab fluid, mucus, sputum, bronchial fluid, culture media, or a fecal or tissue homogenate).

8. The method of claim 1 to 7, wherein said liquid sample comprises milk or colostrum.

9. The method of claim 1 to 8, wherein said at least one first magnet is rectangular, and said magnetically attractable particles are immobilized in a line on the internal wall of said sample container.

10. The method of claim 9, wherein said at least one magnet comprises a pair of magnets spaced apart from one another, and said magnetically attractable particles are immobilized in a pair of lines spaced apart from one another on the internal wall of said sample container.

11. An apparatus (or ''container fixture^") useful in preparing a liquid sample for further analysis, the liquid sample contained in a sample container, said apparatus comprising:

(a) a body having a top portion and a front side wall portion.

(b) at least one receptacle formed in said body and extending downward from said body top portion, said receptacle configured to receive said sample container, and with said receptacle having a back side wall portion; (c) at least one magnet connected to said receptacle back side wall portion; and

(d) optionally, a retention member connected to said body and configured to force said container against said at least one magnet when said container is positioned in said receptacle.

12. The apparatus of claim 1 1. wherein said receptacle is configured so that said sample container is rota table when partially inserted therein.

13. The apparatus of claim 1 1 or 12. wherein said sample container is cylindrical in shape and has a projecting member formed thereon, said apparatus further comprising:

(e) at least one alignment member connected to said body, operativcly associated with said at least one receptacle, and configured to receive said projecting member and restrain said sample container from rotating in said receptacle.

14. The apparatus of claim 1 1 to 13, wherein said at least one receptacle comprises a pair of parallel receptacles.

15. The apparatus of claim 1 1 to 14, wherein said retention member comprises a coil spring, an elastic band, a screw clamp, or a combination thereof.

16. The apparatus of claim 15. wherein said retention member comprises a single retention member configured in cooperation with both of said pair of receptacles.

17. The apparatus of claim 1 1 to 16, wherein said magnet is rectangular and has a length dimension, said receptacle has a length axis, and wherein said magnet length dimension is substantially parallel to said receptacle length axis.

18. The apparatus of claim 1 1 to 17, wherein said magnet is a rare earth magnet.

19. The apparatus of claim 11 to 18, wherein said magnet comprises a pair of magnets connected to each of said at least one receptacle back side wall portion, with said pair of magnets parallel to and spaced apart from one another in each of said at least one receptacles.

20. The apparatus of claim 19, wherein the poles of both members of said pair of magnets in each of said receptacles face in the same direction.

21. The apparatus of claim 20, wherein the North pole of said at least one magnet in one of said pair of receptacles faces in the opposite direction of the North pole of said at least one magnet in the other of said pair of receptacles.

22. A plurality of apparatus of claim 21 magnetically coupled to one another in front-to-back and/or side-to-side arrangement or orientation.