US20190331675A1 - Devices, methods, and kits for multiplexing a fluid sample via fluid sample reuse - Google Patents
Devices, methods, and kits for multiplexing a fluid sample via fluid sample reuse Download PDFInfo
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- US20190331675A1 US20190331675A1 US16/310,088 US201716310088A US2019331675A1 US 20190331675 A1 US20190331675 A1 US 20190331675A1 US 201716310088 A US201716310088 A US 201716310088A US 2019331675 A1 US2019331675 A1 US 2019331675A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
- G01N33/48714—Physical analysis of biological material of liquid biological material by electrical means for determining substances foreign to the organism, e.g. drugs or heavy metals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
- G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
- G01N33/541—Double or second antibody, i.e. precipitating antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
Definitions
- the presently disclosed and claimed inventive concept(s) relate to a device(s) and method(s) for the detection of at least two analytes, said analytes being of different and distinct compositions, present within a small volume of a single fluid sample.
- a multiplexed panel of the various analytes present within the small volume of the single fluid sample is achieved, while mitigating single fluid sample loss/reduction and allowing for re-use of the single fluid sample.
- the currently employed devices and methods require the use of multiple fluid samples and/or aliquots of a single fluid sample for the detection of analytes present in such sample(s).
- Such devices and methods require a large volume of the fluid sample, the collection of multiple fluid samples from a patient in order to achieve analyte detection, and/or result in fluid sample loss/reduction due to multiple aliquots being taken from the fluid sample for analyte detection. Accordingly, a need exists for new and improved devices and methods of high sensitivity that allow a small volume of a single fluid sample obtained from a patient to be re-used in order to detect at least two analytes present in the single fluid sample.
- Such devices and methods thereby allow for the creation of a multiplexed panel associated with the various analytes present in the single fluid sample. It is to such devices and methods, as well as kits related thereto, that the presently disclosed and claimed inventive concept(s) is directed.
- FIGS. 1A, 1B, and 1C are detailed perspective views of one embodiment of a device constructed in accordance with the presently disclosed and claimed inventive concept(s) and use thereof in a method as described herein.
- FIGS. 2A-2L are perspective views of an alternate embodiment of a device of the presently disclosed and claimed inventive concept(s) and use thereof in a method as described herein.
- inventive concept(s) Before explaining at least one embodiment of the inventive concept(s) in detail by way of exemplary drawings, experimentation, results, and laboratory procedures, it is to be understood that the inventive concept(s) is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings, experimentation and/or results.
- inventive concept(s) is capable of other embodiments or of being practiced or carried out in various ways.
- the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary—not exhaustive.
- phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
- compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this presently disclosed and claimed inventive concept(s) have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the presently disclosed and claimed inventive concept(s). All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the inventive concept(s) as defined by the appended claims.
- the designated value may vary by ⁇ 20% or ⁇ 10%, or ⁇ 5%, or ⁇ 1%, or ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art.
- the use of the term “at least one” will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc.
- the term “at least one” may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100/1000 are not to be considered limiting, as higher limits may also produce satisfactory results.
- the terms “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
- A, B, C, or combinations thereof refers to all permutations and combinations of the listed items preceding the term.
- “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
- expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth.
- BB BB
- AAA AAA
- AAB BBC
- AAABCCCCCC CBBAAA
- CABABB CABABB
- the term “substantially” means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree.
- the term “substantially” means that the subsequently described event or circumstance occurs at least 90% of the time, or at least 95% of the time, or at least 98% of the time.
- association with includes both direct association of two moieties to one another as well as indirect association of two moieties to one another.
- Non-limiting examples of associations include covalent binding of one moiety to another moiety either by a direct bond or through a spacer group, non-covalent binding of one moiety to another moiety either directly or by means of specific binding pair members bound to the moieties, incorporation of one moiety into another moiety such as by dissolving one moiety in another moiety or by synthesis, and coating one moiety on another moiety.
- purified means at least one order of magnitude of purification is achieved compared to the starting material or of material in its natural state, for example but not by way of limitation, two, three, four or five orders of magnitude of purification of the starting material or of the natural material.
- purified does not necessarily mean that the material is 100% purified, and therefore such term does not exclude the presence of other material(s) present in the purified composition.
- sample as used herein will be understood to include any type of biological fluid sample that may be utilized in accordance with the presently disclosed and claimed inventive concept(s).
- biological samples include, but are not limited to, whole blood or any portion thereof (i.e., plasma or serum), saliva, sputum, cerebrospinal fluid (CSF), intestinal fluid, intraperotineal fluid, cystic fluid, sweat, interstitial fluid, tears, mucus, urine, bladder wash, semen, combinations, and the like.
- the volume of the sample utilized in accordance with the presently disclosed and claimed inventive concept(s) is from about 1 to about 100 microliters.
- small volume as it relates to the sample utilized in accordance with the presently disclosed and claimed inventive concept(s) means from about 0.1 microliter to about 90 microliters, or from about 1 microliter to about 75 microliters, or from about 2 microliters to about 60 microliters, or less than or equal to about 50 microliters.
- a patient includes human and veterinary subjects.
- a patient is a mammal.
- the patient is a human.
- “Mammal” for purposes of treatment refers to any animal classified as a mammal, including human, domestic and farm animals, nonhuman primates, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.
- the kit can further include a set of written or pictorial instructions (or information on how to obtain instructions, either written or pictorial, from the internet) explaining how to use the kit.
- a kit of this nature can be used in any of the methods described or otherwise contemplated herein.
- reagents used in the fields of biological, chemical, or biochemical analyses could be used in the devices, kits, and methods of the presently claimed and disclosed inventive concept(s). It is contemplated that these reagents may undergo physical and/or chemical changes when bound to an analyte of interest whereby the intensity, nature, frequency, or type of signal generated by the reagent-analyte complex is proportional to the concentration of the analyte existing within the fluid sample. These reagents may contain indicator dyes, metal, enzymes, polymers, antibodies, and electrochemically reactive ingredients.
- any method of detecting and measuring the analyte in a fluid sample can be used in the devices, kits, and methods of the presently claimed and inventive concepts.
- assays for detecting analytes are well known in the art and include, but are not limited to, enzyme inhibition assays, antibody stains, latex agglutination, and immunoassays, such as, radioimmunoassays.
- antibody herein is used in the broadest sense and refers to, for example, intact monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), and to antibody fragments that exhibit the desired biological activity (e.g., antigen/analyte-binding).
- the antibody can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or sub-class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).
- IgG IgG, IgE, IgM, IgD, and IgA
- sub-class e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2
- binding partner as used herein will be understood to refer to any molecule capable of associating with another molecule.
- the binding partner may be an antibody (including polyclonal or monoclonal antibodies), antibody fragments (such as, but not limited to, Fab, Fab′, F(ab′)2, Fv, scFv, Fd, diabodies, single-chain antibodies, and other antibody fragments that retain at least a portion of the variable region of the intact antibody), a receptor, a ligand, aptamers, antibody substitute proteins or peptides (i.e., engineered binding proteins/peptides), molecular imprinted polymers (i.e., inorganic matrices), combinations or derivatives thereof, as well as any other molecules capable of binding to the analyte(s).
- an antibody including polyclonal or monoclonal antibodies
- antibody fragments such as, but not limited to, Fab, Fab′, F(ab′)2, Fv, scFv, Fd, di
- non-reacted portion as used herein will be understood to refer to any portion of the single fluid sample in which an analyte has not become associated with an immobilized capture antibody/binding partner present on an inner surface of a pipette and which is re-dispensed into the sample receptacle.
- reaction portion as used herein will be understood to refer to any portion of the single fluid sample in which an analyte has become associated with an immobilized capture antibody/binding partner on an inner surface of a pipette and which is not re-dispensed into the sample receptacle.
- immunoassays are not strictly limited to immunoassays and may include, by way of example and not by limitation, nucleic acid capture assays and serology-based assays.
- Immunoassays including radioimmunoassays and enzyme-linked immunoassays, are useful methods for use with the presently claimed and disclosed inventive concepts.
- a variety of immunoassay formats including, for example, competitive and non-competitive immunoassay formats, antigen/analyte capture assays and two-antibody sandwich assays can be used in the methods of the invention.
- Enzyme-linked immunosorbent assays can be used in the presently claimed and disclosed inventive concepts, as well.
- an enzyme immunoassay an enzyme is typically conjugated to a second antibody, generally by means of glutaraldehyde, periodate, hetero-bifunctional crosslinking agents, or biotin-streptavidin complexes.
- the analytes are detected and measured using chemiluminescent detection.
- analyte-specific capture antibodies are used to capture an analyte present in the biological fluid sample.
- the analyte-specific capture antibodies (and/or the specific analyte(s)) may be labeled with a chemiluminescent detection antibody to thereby detect if an analyte is present in the biological fluid sample.
- Any chemiluminescent label and detection system can be used in the present devices and methods.
- Chemiluminescent detection antibodies/binding partners can be obtained commercially from various sources. Methods of detecting chemiluminescent detection antibodies/binding partners are known in the art and are not further discussed here in detail.
- Fluorescent detection also can be useful for detecting analytes in the presently claimed and disclosed inventive concepts.
- Useful fluorochromes include, but are not limited to, DAPI, fluorescein, lanthanide metals, Hoechst 33258, R-phycocuanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red and lissamine. Fluorescent compounds can be chemically coupled to antibodies/binding partners without altering their binding capacity.
- Radioimmunoassays can also be useful in certain methods of the invention. Such assays are well known in the art. Radioimmunoassays can be performed, for example, with 125 I-labeled detection antibodies/binding partners that are associated with either the analyte being detected and/or the immobilized capture antibodies/binding partners.
- Assays including, but not limited to, immunoassays, nucleic acid capture assays, and serology-based assays, can be developed for a multiplexed panel of proteins, peptides, and nucleic acids which may be contained within a fluid sample, with such proteins and peptides including, for example but not by way of limitation, albumin, hemoglobin, myoglobin, ⁇ -1-microglobin, immunoglobins, enzymes, glycoproteins, protease inhibitors, drugs, and cytokines.
- the device may be used for the analysis of any fluid sample, including, without limitation, whole blood, plasma, serum, or urine.
- Table 1 below, by way of example only and not by way of limitation, illustrates particular analytes of interest as well as respective antibodies/binding partners which may be used in the presently disclosed and claimed inventive concept(s).
- NA IgG serum EBV Antibody Panel
- VCA IgG serum EBV Antibody Panel
- VCA IgM serum EBV Antibody Panel
- VCA IgM serum Ecstasy (MDMA) Endomysial Antibody
- EMA IgA - serum ELISA and IFA
- EBV Antibody Panel EMA IgG - serum (ELISA and IFA) Estradiol - serum Estriol - serum/plasma Estriol - urine (24 hour collection)
- Ferritin - serum Fibrinogen - plasma Folate - serum Follicle Stimulating Hormone - serum/plasma Follicle Stimulating Hormone - urine Gastrin - serum Gentamicin - serum Growth Hormone - serum H.
- any binding partner may be immobilized on the inner surface(s) of the pipette(s) to accomplish the inventive concept(s), including, by way of example and not by limitation, nucleic acids and fragments thereof, antigens, ligands, and any binding partner capable of accomplishing the presently disclosed and claimed inventive concept(s).
- FIGS. 1A-1C shown therein is an exemplary embodiment of a device 10 that detects the presence and/or absence of at least two analytes in a single fluid sample obtained from a patient.
- the device 10 comprises a sample receptacle 12 and at least one pipette 14 , i.e., the device 10 may contain 1, 2, 5, 10, 15, 20, 50, 100, 100 or 1000 pipettes attached to one another via a mechanical connecting web or a pipetting station, such as pipetting station 15 for example, but not by way of limitation.
- FIGS. 1A-1C depict two pipettes shown as 14 and 26 to illustrate that the device 10 may comprise more than one pipette.
- Pipette 14 comprises an inner surface 16 on which at least one immobilized capture antibody 20 is present.
- the sample receptacle 12 contains a single fluid sample 18 that has been obtained from a patient.
- five immobilized capture antibodies 20 are shown as being immobilized on the inner surface 16 .
- any number of immobilized capture antibodies 20 may be immobilized on the inner surface 16 of the pipette 14 .
- the single fluid sample 18 has been withdrawn into the pipette 14 such that the single fluid sample 18 is in contact with the inner surface 16 of the pipette 14 .
- the single fluid sample 18 comes into contact with immobilized capture antibody/binding partner 20 .
- immobilized capture antibody/binding partner 20 If an analyte of interest is present in the single fluid sample 18 , at least one analyte-antibody/binding partner complex 24 is formed by an association between the analyte 22 present in the single fluid sample 18 and the at least one immobilized capture antibody/binding partner 20 present on the inner surface 16 of the pipette 14 .
- a non-reacted portion of the single fluid sample 18 is re-dispensed into the sample receptacle 12 from the pipette 14 .
- the analyte 22 if present, remains within the pipette 14 (a reacted portion of the single fluid sample 18 ) due to the association between the analyte 22 and the immobilized capture antibody 20 forming the first analyte-antibody/binding partner complex 24 present on the inner surface 16 of the pipette 14 .
- Detection of the analyte 22 within the pipette 14 can be accomplished in accordance with any of the methodologies described herein or well known in the art. The detection can be either a qualitative or quantitative assessment of the presence of analyte 22 within the pipette 14 and thereby indicates the presence of analyte 22 in the single fluid sample 18 .
- pipette 14 is removed from the sample receptacle 12 and pipette 26 is placed within the sample receptacle 12 . Thereafter, at least a portion of the non-reacted portion of the single fluid sample 18 is withdrawn into pipette 26 and into contact with an inner surface 28 of pipette 26 to which a second immobilized capture antibody 30 is bound.
- a second analyte (not shown) which is different in composition than the analyte 22 is thereafter, if present in the single fluid sample 18 , bound to the second immobilized capture antibody/binding partner 30 bound to inner surface 28 of pipette thereby forming a second analyte-antibody/binding partner complex (not shown).
- FIGS. 2A-2L shown therein is an alternate embodiment of a device 10 A that detects the presence and/or absence of at least two analytes in a single fluid sample obtained from a patient.
- the device 10 A comprises a sample receptacle 12 A, a first pipette 14 A, a second pipette 26 A, and a third pipette 36 A. While the non-limiting embodiment of device 10 A as shown in FIGS.
- 2A-2L is shown as comprising three pipettes, three analytes, and three distinct and different immobilized capture antibodies/binding partners, it should be understood to a person having ordinary skill in the art that device 10 A can be comprised of any number of pipettes, analytes, and different and distinct immobilized capture antibodies/binding partners in order to detect the presence of at least two analytes present within a single fluid sample obtained from a patient.
- the device 10 A may contain 1, 2, 5, 10, 15, 20, 50, 100, or 1000 pipettes.
- the pipettes may be attached to one another via a mechanical connecting web or pipetting station, such as pipetting station 15 A for example, but not by way of limitation.
- the process of sequentially disposing the first pipette 14 A, the second pipette 26 A, and the third pipette 36 A within the sample receptacle 12 A can be accomplished either by a manual or automated process(es) via use of the pipetting station 15 A.
- the first pipette 14 A comprises an inner surface 16 A on which a first immobilized capture antibody 20 A is present. As shown in FIGS. 2A-2D , five first immobilized capture antibodies/binding partners 20 A are shown as being immobilized on the inner surface 16 A. However, one of ordinary skill in the art would recognize that any number of first immobilized capture antibodies/binding partners 20 A may be immobilized on the inner surface 16 A of the first pipette 14 A.
- the first pipette 14 A is disposed within sample receptacle 12 A which contains a single fluid sample 18 A obtained from a patient, said single fluid sample 18 A comprising at least two analytes.
- At least a portion of the single fluid sample 18 A is withdrawn into the first pipette 14 A such that the single fluid sample 18 A is in contact with the inner surface 16 A of the first pipette 14 A. As a result of this contact, the single fluid sample 18 A comes into contact with the first immobilized capture antibody/binding partner 20 A. If a first analyte of interest is present in the single fluid sample 18 A, at least one first analyte-antibody/binding partner complex 24 A is formed by an association between the first analyte 22 A present in the single fluid sample 18 A and the first immobilized capture antibody 20 A present on the inner surface 16 A of the first pipette 14 A.
- a non-reacted portion of the single fluid sample 18 A is re-dispensed into the sample receptacle 12 A from the first pipette 14 A.
- the first analyte 22 A if present, remains within the first pipette 14 A (a reacted portion of the single fluid sample 18 A) due to the association between the first analyte 22 A and the first immobilized capture antibody/binding partner 20 A forming the first analyte-antibody complex 24 A present on the inner surface 16 A of the first pipette 14 A.
- Detection of the first analyte 22 A within the first pipette 14 A can be accomplished in accordance with any of the methodologies described herein or well known in the art.
- the detection can be either a qualitative or quantitative assessment of the presence of the first analyte 22 A within the pipette 14 A and thereby indicative of the presence of the first analyte 22 A in the single fluid sample 18 A.
- the second pipette 26 A comprises an inner surface 28 A on which a second immobilized capture antibody/binding partner 30 A is present.
- the second immobilized capture antibody/binding partner 30 A is different in analyte specificity from the first immobilized capture antibody/binding partner 20 A, thereby allowing for association with and detection of a different and distinct analyte present in the single fluid sample 18 A from the first analyte 22 A present in the single fluid sample 18 A.
- the second pipette 26 A is disposable within sample receptacle 12 A which contains the non-reacted portion of the single fluid sample 18 A which has been re-dispensed into the sample receptacle 12 A from the first pipette 14 A.
- FIGS. 2E-2H with respect to the second pipette 26 A, five second immobilized capture antibodies/binding partners 30 A are shown as being immobilized in the inner surface 28 A.
- any number of second immobilized capture antibodies/binding partners 30 A may be immobilized on the inner surface 28 A of the second pipette 26 A.
- At least a portion of the non-reacted portion of the single fluid sample 18 A is withdrawn into the second pipette 26 A such that the single fluid sample 18 A is in contact with the inner surface 28 A of the second pipette 26 A. As a result of this contact, the single fluid sample 18 A comes into contact with the second immobilized capture antibody/binding partner 30 A. If a second analyte of interest is present in the single fluid sample 18 A, at least one second analyte-antibody/binding partner complex 34 A is formed by an association between the second analyte 32 A present in the single fluid sample 18 A and the second immobilized capture antibody/binding 30 A present on the inner surface 28 A of the second pipette 26 A.
- a non-reacted portion of the single fluid sample 18 A is re-dispensed into the sample receptacle 12 A from the second pipette 26 A.
- the second analyte 32 A if present, remains within the second pipette 26 A (a reacted portion of the single fluid sample 18 A) due to the association between the second analyte 32 A and the second immobilized capture antibody/binding partner 30 A forming the second analyte-antibody/binding partner complex 34 A present on the inner surface 28 A of the second pipette 26 A. Detection of the second analyte 32 A within the second pipette 26 A can be accomplished in accordance with any of the methodologies described herein or well known in the art.
- the third pipette 36 A is transitioned so that it is capable of being disposed within the sample receptacle 12 A.
- the third pipette 36 A comprises an inner surface 38 A on which a third immobilized capture antibody/binding partner 40 A is present.
- the third immobilized capture antibody/binding partner 40 A is different in analyte specificity from the first immobilized capture antibody/binding partner 20 A and the second immobilized capture antibody/binding partner 30 A, thereby allowing for association with and detection of a different and distinct analyte present in the single fluid sample 18 A from the first analyte 22 A and second analyte 32 A present in the single fluid sample 18 A.
- the third pipette 36 A is disposed within sample receptacle 12 A which contains the non-reacted portion of the single fluid sample 18 A which has been re-dispensed into the sample receptacle 12 A from the second pipette 26 A.
- sample receptacle 12 A contains the non-reacted portion of the single fluid sample 18 A which has been re-dispensed into the sample receptacle 12 A from the second pipette 26 A.
- five third immobilized capture antibodies/binding partners 40 A are shown as being immobilized on the inner surface 38 A.
- any number of third immobilized capture antibodies/binding partners 40 A may be immobilized on the inner surface 38 A of the third pipette 36 A.
- At least a portion of the non-reacted portion of the single fluid sample 18 A is withdrawn into the third pipette 36 A such that the single fluid sample 18 A is in contact with the inner surface 38 A of the third pipette 36 A. As a result of this contact, the single fluid sample 18 A comes into contact with the third immobilized capture antibody/binding partner 40 A. If a third analyte of interest is present in the single fluid sample 18 A, at least one third analyte-antibody complex 44 A is formed by an association between the third analyte 42 A present in the single fluid sample 18 A and the third immobilized capture antibody/binding partner 40 A present on the inner surface 38 A of the third pipette 36 A.
- a non-reacted portion of the single fluid sample 18 A is re-dispensed into the sample receptacle 12 A from the third pipette 36 A.
- the third analyte 42 A if present, remains within the third pipette 36 A (a reacted portion of the single fluid sample 18 A) due to the association between the third analyte 42 A and the third immobilized capture antibody/binding partner 40 A forming the third analyte-antibody complex 44 A present on the inner surface 38 A of the third pipette 36 A.
- Detection of the third analyte 42 A within the third pipette 36 A can be accomplished in accordance with any of the methodologies described herein or well known in the art. The detection can be either a qualitative or quantitative assessment of the presence of the third analyte 42 A within the third pipette 36 A and thereby indicative of the presence of the third analyte 42 A in the single fluid sample 18 A.
- a device for detecting the presence of at least two analytes in a fluid sample comprising a pipetting station, wherein the pipetting station is configured to receive at least two pipettes, wherein the at least two pipettes comprises a first pipette having a first immobilized binding partner specific to a first analyte present on an inner surface of the first pipette and a second pipette having a second immobilized binding partner specific to a second analyte present on an inner surface of the second pipette, wherein the analyte specificity of the first immobilized binding partner is different than the analyte specificity of the second immobilized binding partner; wherein the first pipette is disposable within a fluid sample receptacle configured to hold a fluid sample, the first pipette configured to withdraw at least a portion of the single fluid sample into the first pipette such that the first immobilized binding partner is brought into contact with the withdrawn fluid sample to thereby form a first analy
- the fluid sample of the device is selected from the group consisting of whole blood, blood plasma, blood serum, saliva, sputum, cerebrospinal fluid (CSF), intestinal fluid, intraperotineal fluid, cystic fluid, sweat, interstitial fluid, tears, mucus, urine, bladder wash, and semen.
- CSF cerebrospinal fluid
- the fluid sample of the device comprises a volume from about 1 microliter to about 100 microliters.
- the volume of the fluid sample of the device is about 50 microliters.
- the sample receptacle of the device is selected from the group consisting of a well, cuvette, tube, vial, and capillary.
- the first analyte and second analyte of the device are selected from the group consisting of cardiac markers, infectious disease serological analytes, therapeutic drugs, drugs of abuse, hormones, cancer markers, infectious disease nucleic acid analytes, autoimmune disease nucleic acid markers, proteins, vitamins, cofactors, metabolites, and combinations thereof.
- the first immobilized binding partner and the second immobilized binding partner of the device are selected from the group consisting of intact monoclonal antibodies, polyclonal antibodies, multi-specific antibodies, bispecific antibodies, antibody fragments, receptors, ligands, aptamers, antigens, antibody substitute proteins or peptides, molecular imprinted polymers, and combinations thereof.
- the first immobilized capture antibody and the second immobilized capture antibody of the device are selected from the group consisting of IgG, IgE, IgM, IgD, IgA, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and combinations thereof.
- a method for detecting the presence of at least two analytes in a fluid sample comprising the steps of upon receipt of a fluid sample which may comprise an analyte, placing the single fluid sample in a sample receptacle; drawing the single fluid sample into a first pipette that is disposed within the sample receptacle, wherein a first immobilized binding partner is present on the an inner surface of the first pipette and the first immobilized binding partner is brought into contact with the fluid sample such that a first analyte-binding partner complex is formed if a first analyte is present in the single fluid sample; re-dispensing a non-reacted portion of the fluid sample from the first pipette and into the fluid sample receptacle; drawing the non-reacted portion of the fluid sample into a second pipette that is disposed within the sample receptacle, wherein a second immobilized binding partner having an analyte specificity different than the first immobilized
- the fluid sample of the method is selected from the group consisting of whole blood, blood plasma, blood serum, saliva, sputum, cerebrospinal fluid (CSF), intestinal fluid, intraperotineal fluid, cystic fluid, sweat, interstitial fluid, tears, mucus, urine, bladder wash, and semen.
- CSF cerebrospinal fluid
- the fluid sample of the method comprises a volume from about 1 microliter to about 100 microliters.
- the volume of the fluid sample of the device is about 50 microliters.
- the sample receptacle of the method is selected from the group consisting of a well, cuvette, tube, vial, and capillary.
- the first analyte and second analyte of the method are selected from the group consisting of cardiac markers, infectious disease serological analytes, therapeutic drugs, drugs of abuse, hormones, cancer markers, infectious disease nucleic acid analytes, autoimmune disease nucleic acid markers, proteins, vitamins, cofactors, metabolites, and combinations thereof.
- the first immobilized binding partner and the second immobilized binding partner of the method are selected from the group consisting of intact monoclonal antibodies, polyclonal antibodies, multi-specific antibodies, bispecific antibodies, antibody fragments, receptors, ligands, aptamers, antigens, antibody substitute proteins and peptides, molecular imprinted polymers, and combinations thereof.
- the first immobilized binding partner and the second immobilized binding partner of the method are selected from the group consisting of IgG, IgE, IgM, IgD, IgA, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and combinations thereof.
- the detection of the first analyte and the second analyte of the method is conducted via chemiluminescent detection, fluorescent detection, and combinations thereof.
- a kit for detecting the presence of at least two analytes in a fluid sample comprising at least two pipettes, wherein the at least two pipettes comprises a first pipette having a first immobilized capture antibody specific to a first analyte present on an inner surface of the first pipette and a second pipette having a second immobilized capture antibody specific to a second analyte present on an inner surface of the second pipette, wherein the analyte specificity of the first immobilized capture antibody is different than the analyte specificity of the second immobilized capture antibody; a fluid sample receptacle, wherein the fluid sample receptacle is configured to hold a fluid sample; and providing instructions to a user to allow the user to use the kit to identify the presence of at least two analytes in the fluid sample.
Abstract
Description
- This application claims priority to U.S. Provisional Application No. 62/351,530, filed Jun. 17, 2016, the disclosure of which is hereby incorporated by reference in its entirety.
- Not Applicable.
- The presently disclosed and claimed inventive concept(s) relate to a device(s) and method(s) for the detection of at least two analytes, said analytes being of different and distinct compositions, present within a small volume of a single fluid sample. As a result of such detection, a multiplexed panel of the various analytes present within the small volume of the single fluid sample is achieved, while mitigating single fluid sample loss/reduction and allowing for re-use of the single fluid sample.
- Numerous devices and methods exist for detecting analytes that may be present in a fluid sample. However, the currently employed devices and methods require the use of multiple fluid samples and/or aliquots of a single fluid sample for the detection of analytes present in such sample(s). Such devices and methods require a large volume of the fluid sample, the collection of multiple fluid samples from a patient in order to achieve analyte detection, and/or result in fluid sample loss/reduction due to multiple aliquots being taken from the fluid sample for analyte detection. Accordingly, a need exists for new and improved devices and methods of high sensitivity that allow a small volume of a single fluid sample obtained from a patient to be re-used in order to detect at least two analytes present in the single fluid sample. Such devices and methods thereby allow for the creation of a multiplexed panel associated with the various analytes present in the single fluid sample. It is to such devices and methods, as well as kits related thereto, that the presently disclosed and claimed inventive concept(s) is directed.
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FIGS. 1A, 1B, and 1C are detailed perspective views of one embodiment of a device constructed in accordance with the presently disclosed and claimed inventive concept(s) and use thereof in a method as described herein. -
FIGS. 2A-2L are perspective views of an alternate embodiment of a device of the presently disclosed and claimed inventive concept(s) and use thereof in a method as described herein. - Before explaining at least one embodiment of the inventive concept(s) in detail by way of exemplary drawings, experimentation, results, and laboratory procedures, it is to be understood that the inventive concept(s) is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings, experimentation and/or results. The inventive concept(s) is capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary—not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
- Unless otherwise defined herein, scientific and technical terms used in connection with the presently disclosed and claimed inventive concept(s) shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.
- All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this presently disclosed and claimed inventive concept(s) pertains. All patents, published patent applications, and non-patent publications referenced in any portion of this application are herein expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.
- All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this presently disclosed and claimed inventive concept(s) have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the presently disclosed and claimed inventive concept(s). All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the inventive concept(s) as defined by the appended claims.
- As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
- The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a compound” may refer to 1 or more, 2 or more, 3 or more, 4 or more or greater numbers of compounds. The term “plurality” refers to “two or more.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. For example but not by way of limitation, when the term “about” is utilized, the designated value may vary by ±20% or ±10%, or ±5%, or ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art. The use of the term “at least one” will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term “at least one” may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100/1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term “at least one of X, Y and Z” will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y and Z. The use of ordinal number terminology (i.e., “first”, “second”, “third”, “fourth”, etc.) is solely for the purpose of differentiating between two or more items and is not meant to imply any sequence or order or importance to one item over another or any order of addition, for example.
- As used in this specification and claim(s), the terms “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
- The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
- As used herein, the term “substantially” means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree. For example, the term “substantially” means that the subsequently described event or circumstance occurs at least 90% of the time, or at least 95% of the time, or at least 98% of the time.
- As used herein, the phrase “associated with” includes both direct association of two moieties to one another as well as indirect association of two moieties to one another. Non-limiting examples of associations include covalent binding of one moiety to another moiety either by a direct bond or through a spacer group, non-covalent binding of one moiety to another moiety either directly or by means of specific binding pair members bound to the moieties, incorporation of one moiety into another moiety such as by dissolving one moiety in another moiety or by synthesis, and coating one moiety on another moiety.
- The term “purified” as used herein means at least one order of magnitude of purification is achieved compared to the starting material or of material in its natural state, for example but not by way of limitation, two, three, four or five orders of magnitude of purification of the starting material or of the natural material. Thus, the term “purified” as utilized herein does not necessarily mean that the material is 100% purified, and therefore such term does not exclude the presence of other material(s) present in the purified composition.
- The term “sample” as used herein will be understood to include any type of biological fluid sample that may be utilized in accordance with the presently disclosed and claimed inventive concept(s). Examples of biological samples that may be utilized include, but are not limited to, whole blood or any portion thereof (i.e., plasma or serum), saliva, sputum, cerebrospinal fluid (CSF), intestinal fluid, intraperotineal fluid, cystic fluid, sweat, interstitial fluid, tears, mucus, urine, bladder wash, semen, combinations, and the like. The volume of the sample utilized in accordance with the presently disclosed and claimed inventive concept(s) is from about 1 to about 100 microliters. As used herein, the term “small volume” as it relates to the sample utilized in accordance with the presently disclosed and claimed inventive concept(s) means from about 0.1 microliter to about 90 microliters, or from about 1 microliter to about 75 microliters, or from about 2 microliters to about 60 microliters, or less than or equal to about 50 microliters.
- The term “patient” includes human and veterinary subjects. In certain embodiments, a patient is a mammal. In certain other embodiments, the patient is a human. “Mammal” for purposes of treatment refers to any animal classified as a mammal, including human, domestic and farm animals, nonhuman primates, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.
- Turning now to particular embodiments of the presently claimed and disclosed inventive concept(s), devices, methods, and kits are herein disclosed of high sensitivity that allow for a small volume of a single fluid sample obtained from a patient to be re-used in order to detect at least two analytes present in the single fluid sample.
- The kit can further include a set of written or pictorial instructions (or information on how to obtain instructions, either written or pictorial, from the internet) explaining how to use the kit. A kit of this nature can be used in any of the methods described or otherwise contemplated herein.
- It is contemplated that virtually any reagent used in the fields of biological, chemical, or biochemical analyses could be used in the devices, kits, and methods of the presently claimed and disclosed inventive concept(s). It is contemplated that these reagents may undergo physical and/or chemical changes when bound to an analyte of interest whereby the intensity, nature, frequency, or type of signal generated by the reagent-analyte complex is proportional to the concentration of the analyte existing within the fluid sample. These reagents may contain indicator dyes, metal, enzymes, polymers, antibodies, and electrochemically reactive ingredients.
- Any method of detecting and measuring the analyte in a fluid sample can be used in the devices, kits, and methods of the presently claimed and inventive concepts. A variety of assays for detecting analytes are well known in the art and include, but are not limited to, enzyme inhibition assays, antibody stains, latex agglutination, and immunoassays, such as, radioimmunoassays. The term “antibody” herein is used in the broadest sense and refers to, for example, intact monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), and to antibody fragments that exhibit the desired biological activity (e.g., antigen/analyte-binding). The antibody can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or sub-class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).
- The term “binding partner” as used herein will be understood to refer to any molecule capable of associating with another molecule. For example, but not by way of limitation, the binding partner may be an antibody (including polyclonal or monoclonal antibodies), antibody fragments (such as, but not limited to, Fab, Fab′, F(ab′)2, Fv, scFv, Fd, diabodies, single-chain antibodies, and other antibody fragments that retain at least a portion of the variable region of the intact antibody), a receptor, a ligand, aptamers, antibody substitute proteins or peptides (i.e., engineered binding proteins/peptides), molecular imprinted polymers (i.e., inorganic matrices), combinations or derivatives thereof, as well as any other molecules capable of binding to the analyte(s).
- The term “non-reacted portion” as used herein will be understood to refer to any portion of the single fluid sample in which an analyte has not become associated with an immobilized capture antibody/binding partner present on an inner surface of a pipette and which is re-dispensed into the sample receptacle. The term “reacted portion” as used herein will be understood to refer to any portion of the single fluid sample in which an analyte has become associated with an immobilized capture antibody/binding partner on an inner surface of a pipette and which is not re-dispensed into the sample receptacle.
- While immunoassays are primarily discussed herein, a person having ordinary skill in the art should readily understand that the presently disclosed and claimed inventive concept(s) are not strictly limited to immunoassays and may include, by way of example and not by limitation, nucleic acid capture assays and serology-based assays. Immunoassays, including radioimmunoassays and enzyme-linked immunoassays, are useful methods for use with the presently claimed and disclosed inventive concepts. A variety of immunoassay formats, including, for example, competitive and non-competitive immunoassay formats, antigen/analyte capture assays and two-antibody sandwich assays can be used in the methods of the invention. Enzyme-linked immunosorbent assays (ELISAs) can be used in the presently claimed and disclosed inventive concepts, as well. In the case of an enzyme immunoassay, an enzyme is typically conjugated to a second antibody, generally by means of glutaraldehyde, periodate, hetero-bifunctional crosslinking agents, or biotin-streptavidin complexes. As will be readily recognized, however, a wide variety of different conjugation techniques exist which are readily available for use with the presently disclosed and claimed inventive concept(s) to one skilled in the art.
- In certain embodiments, the analytes are detected and measured using chemiluminescent detection. For example, in certain embodiments, analyte-specific capture antibodies are used to capture an analyte present in the biological fluid sample. The analyte-specific capture antibodies (and/or the specific analyte(s)) may be labeled with a chemiluminescent detection antibody to thereby detect if an analyte is present in the biological fluid sample. Any chemiluminescent label and detection system can be used in the present devices and methods. Chemiluminescent detection antibodies/binding partners can be obtained commercially from various sources. Methods of detecting chemiluminescent detection antibodies/binding partners are known in the art and are not further discussed here in detail.
- Fluorescent detection also can be useful for detecting analytes in the presently claimed and disclosed inventive concepts. Useful fluorochromes include, but are not limited to, DAPI, fluorescein, lanthanide metals, Hoechst 33258, R-phycocuanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red and lissamine. Fluorescent compounds can be chemically coupled to antibodies/binding partners without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody/binding partner absorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light as a characteristic color that is visually detectable. Radioimmunoassays (RIAs) can also be useful in certain methods of the invention. Such assays are well known in the art. Radioimmunoassays can be performed, for example, with 125I-labeled detection antibodies/binding partners that are associated with either the analyte being detected and/or the immobilized capture antibodies/binding partners.
- Assays, including, but not limited to, immunoassays, nucleic acid capture assays, and serology-based assays, can be developed for a multiplexed panel of proteins, peptides, and nucleic acids which may be contained within a fluid sample, with such proteins and peptides including, for example but not by way of limitation, albumin, hemoglobin, myoglobin, α-1-microglobin, immunoglobins, enzymes, glycoproteins, protease inhibitors, drugs, and cytokines. The device may be used for the analysis of any fluid sample, including, without limitation, whole blood, plasma, serum, or urine. Table 1 below, by way of example only and not by way of limitation, illustrates particular analytes of interest as well as respective antibodies/binding partners which may be used in the presently disclosed and claimed inventive concept(s).
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TABLE 1 Non-Limiting Examples of Analytes and Antibodies/Binding Partners 25-hydroxyvitamin D; 1,25 dihydroxyvitamin D - serum/plasma Acetaminophen - serum/plasma ACTH - serum/plasma AFP - serum ALB - serum/plasma Alpha-1-Antitrypsin blood SERPINA 1 Alpha-1-Antitrypsin serum/plasma Alpha-1-Acid Glycoprotein serum/plasma Amphetamines - urine Amylase - pancreatitis blood, urine or peritioneal sample ANA - autoimmune (SLE) serum/plasma Androstenedione serum/plasma Anti-double-stranded DNA, IgG -serum Apolipoprotein A-I - serum/plasma Barbituates - urine Benzodazepines - serum/plasma Benzodazepines - urine Beta2 Microglobulin - serum or 24 h urine Blood Typing (Immunoassay) - Panel/Whole Blood BNP - plasma Borrelia burgdorferi Antibodies (Lyme Disease) - serum/plasma CA -125 - serum/plasma CA 15-3 - serum/plasma CA 19-9 - serum/plasma CA 27.29 - serum/plasma Calcitonin - serum/plasma Carbamazepine - serum/plasma Cardiac-specific Troponin I and Troponin T - serum/plasma Cardiolipin Antibodies (IgG) - serum/plasma CEA - serum/plasma Celiac Panel (4 plex) - serum/plasma Chlamydia trachomatis DNA Qualitative Chlamydia trachomatis DNA Qualitative - Swab Collection Chlamydia Trachomatis, DNA, Qualitative/Gonorrhea, DNA, Qualitative Cholinesterase - serum/plasma CKMB - serum/plasma CMV Antibody IgG - serum/plasma CMV Antibody IgM - serum/plasma Cocaine - urine Collagen Crosslinks - serum/plasma Cortisol, Total - serum/plasma C-peptide (insulin) - serum/plasma CRP -serum/plasma Cyclic Citrullinated Peptide (CCP) Antibody, IgG - serum/plasma Cyclosporine, A (immunosupressant) - serum/plasma Cystatin C (kidney) - serum/plasma D-Dimer -serum/plasma Deaminated Gliadin Peptide IgA -serum/plasma Deaminated Gliadin Peptide IgG -serum/plasma Dehydroepiandrosterone Sulfate - serum/plasma Dehydroepiandrosterone Sulfate - serum/plasma GC/MS Digoxin - serum/plasma TDM Dolophine (Methadone) Double Stranded DNA (Crithidia luciliae) Antibody, IgG E. coli Shiga Toxin EBV Antibody (NA IgG) - serum EBV Antibody Panel (NA IgG; VCA IgG; VCA IgM) - serum EBV Antibody Panel (VCA IgG) - serum EBV Antibody Panel (VCA IgM) - serum Ecstasy (MDMA) Endomysial Antibody; EMA IgA - serum (ELISA and IFA) Endomysial Antibody; EMA IgG - serum (ELISA and IFA) Estradiol - serum Estriol - serum/plasma Estriol - urine (24 hour collection) Ferritin - serum Fibrinogen - plasma Folate - serum Follicle Stimulating Hormone - serum/plasma Follicle Stimulating Hormone - urine Gastrin - serum Gentamicin - serum Growth Hormone - serum H. pylori, IgG - serum Haptoglobin, Quantitative - serum hCG - serum/plasma hCG - urine Hepatitis A Antibody (HAAb), Total - serum Hepatitis A, IgM - serum Hepatitis B Core Antibody, IgM - serum Hepatitis B Core Antibody, Total - serum Hepatitis B Surface Antibody, Total - serum Hepatitis B, DNA, Quantitative Hepatitis C Antibody - serum Hepatitis C Genotyping HER2/neu, Quantitative - serum Herpes Simplex Virus, Type 1, IgG - serum HIV-1/2 Ab - serum Homocysteine - serum/plasma hsCRP - serum/plasma Human Immunodeficiency Virus 1, RNA Viral Load, Quantitative Human Immunodeficiency Virus Antibody Test - serum IgA, serum IgE IgG, serum IgM, serum Insulin - serum/plasma Lactate Dehydrogenase, Total and Isoenzymes - Body Fluid/Serum Lipase - serum Luteinizing Hormone - serum Luteinizing Hormone - urine Measles (Rubeola Antibody), IgG - serum/CSF Methadone Metabolite - urine Microalbumin - urine Mumps Antibody, IgG - serum Myoglobin - serum/plasma Neisseria gonorrhoeae, DNA, Qualitative Nuclear Antigen Antibody, Jo-1 - serum Nuclear Antigen Antibody, RNP - serum Nuclear Antigen Antibody, Scl-70 - serum Nuclear Antigen Antibody, Sm - serum Nuclear Antigen Antibody, SSA - serum Nuclear Antigen Antibody, SSB - serum Opiates - urine Parathyroid Hormone - serum Phencyclidine (PCP) - urine Phenobarbital - serum/plasma Phenytoin, Total - serum/plasma Progesterone - serum Prolactin - serum Propoxyphene PSA, Free - serum/plasma PSA, Total - serum RBC Antibody ID (Immunoassay) - Whole Blood Rheumatoid Factor, Total - serum Rubella Antibodies, IgG - serum Rubella Antibodies, IgM - serum Salicylate Sex Hormone-binding Globulin (SHBG) Sex Hormone-binding Globulin (SHBG) - serum/plasma Streptolysin O Antibody, Titer (ASO) - serum Syphilis Screen (Treponema Pallidum Antibody) - serum T3, Free - Triiodothyronine, Free - serum T3, Total - Triiodothyronine, Total - serum T4, Free - Thyroxine, Free - serum T4, Total - Thyroxine, Total - serum Testosterone, Free - serum/plasma Testosterone, Total - serum/plasma THC - urine Theophylline - serum Thyroglobulin - serum or plasma Thyroglobulin Antibodies - serum Thyroid Peroxidase Antibody (TPO) Thyroid Stimulating Hormone (TSH) - serum Thyroxine Binding Globulin - serum Tobramycin -serum/plasma Toxoplasma, IgG - serum Toxoplasma, IgM - serum Tricyclic Antidepressants - urine Tricyclic Antidepressants, Blood Triiodothyronine Uptake (T3 Uptake) - serum Valproic Acid - serum/plasma Vancomycin - serum Varicella-Zoster Antibody - serum Viral Hepatitis C, RNA, Quantitative Vitamin B12 - serum - In addition and as illustrated in Table 1 above, while the Figures specifically show immobilized capture antibodies present on the inner surface(s) of the pipette(s), it should be readily understood by a person having ordinary skill in the art that any binding partner may be immobilized on the inner surface(s) of the pipette(s) to accomplish the inventive concept(s), including, by way of example and not by limitation, nucleic acids and fragments thereof, antigens, ligands, and any binding partner capable of accomplishing the presently disclosed and claimed inventive concept(s).
- Referring now to the drawings, and more particularly to
FIGS. 1A-1C , shown therein is an exemplary embodiment of adevice 10 that detects the presence and/or absence of at least two analytes in a single fluid sample obtained from a patient. Thedevice 10 comprises asample receptacle 12 and at least onepipette 14, i.e., thedevice 10 may contain 1, 2, 5, 10, 15, 20, 50, 100, 100 or 1000 pipettes attached to one another via a mechanical connecting web or a pipetting station, such aspipetting station 15 for example, but not by way of limitation. By way of example only,FIGS. 1A-1C depict two pipettes shown as 14 and 26 to illustrate that thedevice 10 may comprise more than one pipette. For purposes of clarity only and not by way of limitation, thedevice 10 shown inFIGS. 1A-1C shall be described with reference only to pipette 14.Pipette 14 comprises aninner surface 16 on which at least oneimmobilized capture antibody 20 is present. Thesample receptacle 12 contains asingle fluid sample 18 that has been obtained from a patient. As shown inFIG. 1A with respect to pipette 14, five immobilizedcapture antibodies 20 are shown as being immobilized on theinner surface 16. However, one of ordinary skill in the art would recognize that any number of immobilizedcapture antibodies 20 may be immobilized on theinner surface 16 of thepipette 14. - As depicted in
FIG. 1B , at least a portion of thesingle fluid sample 18 has been withdrawn into thepipette 14 such that thesingle fluid sample 18 is in contact with theinner surface 16 of thepipette 14. As a result of this contact, thesingle fluid sample 18 comes into contact with immobilized capture antibody/bindingpartner 20. If an analyte of interest is present in thesingle fluid sample 18, at least one analyte-antibody/binding partner complex 24 is formed by an association between theanalyte 22 present in thesingle fluid sample 18 and the at least one immobilized capture antibody/bindingpartner 20 present on theinner surface 16 of thepipette 14. - As shown in
FIG. 1C , a non-reacted portion of thesingle fluid sample 18 is re-dispensed into thesample receptacle 12 from thepipette 14. Theanalyte 22, if present, remains within the pipette 14 (a reacted portion of the single fluid sample 18) due to the association between theanalyte 22 and the immobilizedcapture antibody 20 forming the first analyte-antibody/binding partner complex 24 present on theinner surface 16 of thepipette 14. Detection of theanalyte 22 within thepipette 14 can be accomplished in accordance with any of the methodologies described herein or well known in the art. The detection can be either a qualitative or quantitative assessment of the presence ofanalyte 22 within thepipette 14 and thereby indicates the presence ofanalyte 22 in thesingle fluid sample 18. - Thereafter,
pipette 14 is removed from thesample receptacle 12 andpipette 26 is placed within thesample receptacle 12. Thereafter, at least a portion of the non-reacted portion of thesingle fluid sample 18 is withdrawn intopipette 26 and into contact with aninner surface 28 ofpipette 26 to which a secondimmobilized capture antibody 30 is bound. A second analyte (not shown) which is different in composition than theanalyte 22 is thereafter, if present in thesingle fluid sample 18, bound to the second immobilized capture antibody/bindingpartner 30 bound toinner surface 28 of pipette thereby forming a second analyte-antibody/binding partner complex (not shown). - Referring now to
FIGS. 2A-2L , shown therein is an alternate embodiment of adevice 10A that detects the presence and/or absence of at least two analytes in a single fluid sample obtained from a patient. Thedevice 10A comprises asample receptacle 12A, afirst pipette 14A, asecond pipette 26A, and athird pipette 36A. While the non-limiting embodiment ofdevice 10A as shown inFIGS. 2A-2L is shown as comprising three pipettes, three analytes, and three distinct and different immobilized capture antibodies/binding partners, it should be understood to a person having ordinary skill in the art thatdevice 10A can be comprised of any number of pipettes, analytes, and different and distinct immobilized capture antibodies/binding partners in order to detect the presence of at least two analytes present within a single fluid sample obtained from a patient. For example, thedevice 10A may contain 1, 2, 5, 10, 15, 20, 50, 100, or 1000 pipettes. The pipettes may be attached to one another via a mechanical connecting web or pipetting station, such aspipetting station 15A for example, but not by way of limitation. In addition, the process of sequentially disposing thefirst pipette 14A, thesecond pipette 26A, and thethird pipette 36A within thesample receptacle 12A can be accomplished either by a manual or automated process(es) via use of thepipetting station 15A. - As shown in
FIGS. 2A-2D , thefirst pipette 14A comprises aninner surface 16A on which a firstimmobilized capture antibody 20A is present. As shown inFIGS. 2A-2D , five first immobilized capture antibodies/bindingpartners 20A are shown as being immobilized on theinner surface 16A. However, one of ordinary skill in the art would recognize that any number of first immobilized capture antibodies/bindingpartners 20A may be immobilized on theinner surface 16A of thefirst pipette 14A. Thefirst pipette 14A is disposed withinsample receptacle 12A which contains asingle fluid sample 18A obtained from a patient, saidsingle fluid sample 18A comprising at least two analytes. At least a portion of thesingle fluid sample 18A is withdrawn into thefirst pipette 14A such that thesingle fluid sample 18A is in contact with theinner surface 16A of thefirst pipette 14A. As a result of this contact, thesingle fluid sample 18A comes into contact with the first immobilized capture antibody/binding partner 20A. If a first analyte of interest is present in thesingle fluid sample 18A, at least one first analyte-antibody/binding partner complex 24A is formed by an association between thefirst analyte 22A present in thesingle fluid sample 18A and the firstimmobilized capture antibody 20A present on theinner surface 16A of thefirst pipette 14A. A non-reacted portion of thesingle fluid sample 18A is re-dispensed into thesample receptacle 12A from thefirst pipette 14A. Thefirst analyte 22A, if present, remains within thefirst pipette 14A (a reacted portion of thesingle fluid sample 18A) due to the association between thefirst analyte 22A and the first immobilized capture antibody/binding partner 20A forming the first analyte-antibody complex 24A present on theinner surface 16A of thefirst pipette 14A. Detection of thefirst analyte 22A within thefirst pipette 14A can be accomplished in accordance with any of the methodologies described herein or well known in the art. The detection can be either a qualitative or quantitative assessment of the presence of thefirst analyte 22A within thepipette 14A and thereby indicative of the presence of thefirst analyte 22A in thesingle fluid sample 18A. After the non-reacted portion of thesingle fluid sample 18A is re-dispensed into thesample receptacle 12A from thefirst pipette 14A, thesecond pipette 26A is transitioned so that is capable of being disposed within thesample receptacle 12A. - Referring now to
FIGS. 2E-2H , thesecond pipette 26A comprises aninner surface 28A on which a second immobilized capture antibody/binding partner 30A is present. The second immobilized capture antibody/binding partner 30A is different in analyte specificity from the first immobilized capture antibody/binding partner 20A, thereby allowing for association with and detection of a different and distinct analyte present in thesingle fluid sample 18A from thefirst analyte 22A present in thesingle fluid sample 18A. Thesecond pipette 26A is disposable withinsample receptacle 12A which contains the non-reacted portion of thesingle fluid sample 18A which has been re-dispensed into thesample receptacle 12A from thefirst pipette 14A. As shown inFIGS. 2E-2H , with respect to thesecond pipette 26A, five second immobilized capture antibodies/bindingpartners 30A are shown as being immobilized in theinner surface 28A. However, one of ordinary skill in the art would recognize that any number of second immobilized capture antibodies/bindingpartners 30A may be immobilized on theinner surface 28A of thesecond pipette 26A. At least a portion of the non-reacted portion of thesingle fluid sample 18A is withdrawn into thesecond pipette 26A such that thesingle fluid sample 18A is in contact with theinner surface 28A of thesecond pipette 26A. As a result of this contact, thesingle fluid sample 18A comes into contact with the second immobilized capture antibody/binding partner 30A. If a second analyte of interest is present in thesingle fluid sample 18A, at least one second analyte-antibody/binding partner complex 34A is formed by an association between thesecond analyte 32A present in thesingle fluid sample 18A and the second immobilized capture antibody/binding 30A present on theinner surface 28A of thesecond pipette 26A. A non-reacted portion of thesingle fluid sample 18A is re-dispensed into thesample receptacle 12A from thesecond pipette 26A. Thesecond analyte 32A, if present, remains within thesecond pipette 26A (a reacted portion of thesingle fluid sample 18A) due to the association between thesecond analyte 32A and the second immobilized capture antibody/binding partner 30A forming the second analyte-antibody/binding partner complex 34A present on theinner surface 28A of thesecond pipette 26A. Detection of thesecond analyte 32A within thesecond pipette 26A can be accomplished in accordance with any of the methodologies described herein or well known in the art. The detection can be either a qualitative or quantitative assessment of the presence of thesecond analyte 32A within thesecond pipette 26A and thereby indicative of the presence of thesecond analyte 32A in thesingle fluid sample 18A=. After the non-reacted portion of thesingle fluid sample 18A is re-dispensed into thesample receptacle 12A, thethird pipette 36A is transitioned so that it is capable of being disposed within thesample receptacle 12A. - Referring now to
FIGS. 21-2L , thethird pipette 36A comprises aninner surface 38A on which a third immobilized capture antibody/binding partner 40A is present. The third immobilized capture antibody/binding partner 40A is different in analyte specificity from the first immobilized capture antibody/binding partner 20A and the second immobilized capture antibody/binding partner 30A, thereby allowing for association with and detection of a different and distinct analyte present in thesingle fluid sample 18A from thefirst analyte 22A andsecond analyte 32A present in thesingle fluid sample 18A. Thethird pipette 36A is disposed withinsample receptacle 12A which contains the non-reacted portion of thesingle fluid sample 18A which has been re-dispensed into thesample receptacle 12A from thesecond pipette 26A. As shown inFIGS. 21-2L , with respect to thethird pipette 36A, five third immobilized capture antibodies/bindingpartners 40A are shown as being immobilized on theinner surface 38A. However, one of ordinary skill in the art would recognize that any number of third immobilized capture antibodies/bindingpartners 40A may be immobilized on theinner surface 38A of thethird pipette 36A. At least a portion of the non-reacted portion of thesingle fluid sample 18A is withdrawn into thethird pipette 36A such that thesingle fluid sample 18A is in contact with theinner surface 38A of thethird pipette 36A. As a result of this contact, thesingle fluid sample 18A comes into contact with the third immobilized capture antibody/binding partner 40A. If a third analyte of interest is present in thesingle fluid sample 18A, at least one third analyte-antibody complex 44A is formed by an association between the third analyte 42A present in thesingle fluid sample 18A and the third immobilized capture antibody/binding partner 40A present on theinner surface 38A of thethird pipette 36A. A non-reacted portion of thesingle fluid sample 18A is re-dispensed into thesample receptacle 12A from thethird pipette 36A. The third analyte 42A, if present, remains within thethird pipette 36A (a reacted portion of thesingle fluid sample 18A) due to the association between the third analyte 42A and the third immobilized capture antibody/binding partner 40A forming the third analyte-antibody complex 44A present on theinner surface 38A of thethird pipette 36A. Detection of the third analyte 42A within thethird pipette 36A can be accomplished in accordance with any of the methodologies described herein or well known in the art. The detection can be either a qualitative or quantitative assessment of the presence of the third analyte 42A within thethird pipette 36A and thereby indicative of the presence of the third analyte 42A in thesingle fluid sample 18A. - A device for detecting the presence of at least two analytes in a fluid sample, the device comprising a pipetting station, wherein the pipetting station is configured to receive at least two pipettes, wherein the at least two pipettes comprises a first pipette having a first immobilized binding partner specific to a first analyte present on an inner surface of the first pipette and a second pipette having a second immobilized binding partner specific to a second analyte present on an inner surface of the second pipette, wherein the analyte specificity of the first immobilized binding partner is different than the analyte specificity of the second immobilized binding partner; wherein the first pipette is disposable within a fluid sample receptacle configured to hold a fluid sample, the first pipette configured to withdraw at least a portion of the single fluid sample into the first pipette such that the first immobilized binding partner is brought into contact with the withdrawn fluid sample to thereby form a first analyte-binding partner complex if a first analyte is present in the fluid sample; and further wherein a non-reacted portion of the single fluid sample is re-dispensable from the first pipette into the fluid sample receptacle, and thereafter the second pipette is disposable within the fluid sample receptacle whereby the second pipette is configured to withdraw at least a portion of the non-reacted portion of the fluid sample such that the second immobilized binding partner is brought into contact with the non-reacted portion of the withdrawn fluid sample to thereby form a second analyte-binding partner complex if a second analyte is present in the non-reacted portion of the single fluid sample.
- The fluid sample of the device is selected from the group consisting of whole blood, blood plasma, blood serum, saliva, sputum, cerebrospinal fluid (CSF), intestinal fluid, intraperotineal fluid, cystic fluid, sweat, interstitial fluid, tears, mucus, urine, bladder wash, and semen.
- The fluid sample of the device comprises a volume from about 1 microliter to about 100 microliters. The volume of the fluid sample of the device is about 50 microliters.
- The sample receptacle of the device is selected from the group consisting of a well, cuvette, tube, vial, and capillary.
- The first analyte and second analyte of the device are selected from the group consisting of cardiac markers, infectious disease serological analytes, therapeutic drugs, drugs of abuse, hormones, cancer markers, infectious disease nucleic acid analytes, autoimmune disease nucleic acid markers, proteins, vitamins, cofactors, metabolites, and combinations thereof.
- The first immobilized binding partner and the second immobilized binding partner of the device are selected from the group consisting of intact monoclonal antibodies, polyclonal antibodies, multi-specific antibodies, bispecific antibodies, antibody fragments, receptors, ligands, aptamers, antigens, antibody substitute proteins or peptides, molecular imprinted polymers, and combinations thereof.
- The first immobilized capture antibody and the second immobilized capture antibody of the device are selected from the group consisting of IgG, IgE, IgM, IgD, IgA, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and combinations thereof.
- A method for detecting the presence of at least two analytes in a fluid sample, the method comprising the steps of upon receipt of a fluid sample which may comprise an analyte, placing the single fluid sample in a sample receptacle; drawing the single fluid sample into a first pipette that is disposed within the sample receptacle, wherein a first immobilized binding partner is present on the an inner surface of the first pipette and the first immobilized binding partner is brought into contact with the fluid sample such that a first analyte-binding partner complex is formed if a first analyte is present in the single fluid sample; re-dispensing a non-reacted portion of the fluid sample from the first pipette and into the fluid sample receptacle; drawing the non-reacted portion of the fluid sample into a second pipette that is disposed within the sample receptacle, wherein a second immobilized binding partner having an analyte specificity different than the first immobilized binding partner is present on an inner surface of the second pipette and the second immobilized binding partner is brought into contact with the non-reacted portion of the fluid sample such that a second analyte-binding partner complex is formed if a second analyte is present in the non-reacted portion of the fluid sample; and detecting the presence of the first analyte-binding partner complex and the presence of the second analyte-binding partner complex.
- The fluid sample of the method is selected from the group consisting of whole blood, blood plasma, blood serum, saliva, sputum, cerebrospinal fluid (CSF), intestinal fluid, intraperotineal fluid, cystic fluid, sweat, interstitial fluid, tears, mucus, urine, bladder wash, and semen.
- The fluid sample of the method comprises a volume from about 1 microliter to about 100 microliters. The volume of the fluid sample of the device is about 50 microliters.
- The sample receptacle of the method is selected from the group consisting of a well, cuvette, tube, vial, and capillary.
- The first analyte and second analyte of the method are selected from the group consisting of cardiac markers, infectious disease serological analytes, therapeutic drugs, drugs of abuse, hormones, cancer markers, infectious disease nucleic acid analytes, autoimmune disease nucleic acid markers, proteins, vitamins, cofactors, metabolites, and combinations thereof.
- The first immobilized binding partner and the second immobilized binding partner of the method are selected from the group consisting of intact monoclonal antibodies, polyclonal antibodies, multi-specific antibodies, bispecific antibodies, antibody fragments, receptors, ligands, aptamers, antigens, antibody substitute proteins and peptides, molecular imprinted polymers, and combinations thereof.
- The first immobilized binding partner and the second immobilized binding partner of the method are selected from the group consisting of IgG, IgE, IgM, IgD, IgA, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and combinations thereof.
- The detection of the first analyte and the second analyte of the method is conducted via chemiluminescent detection, fluorescent detection, and combinations thereof.
- A kit for detecting the presence of at least two analytes in a fluid sample, the kit comprising at least two pipettes, wherein the at least two pipettes comprises a first pipette having a first immobilized capture antibody specific to a first analyte present on an inner surface of the first pipette and a second pipette having a second immobilized capture antibody specific to a second analyte present on an inner surface of the second pipette, wherein the analyte specificity of the first immobilized capture antibody is different than the analyte specificity of the second immobilized capture antibody; a fluid sample receptacle, wherein the fluid sample receptacle is configured to hold a fluid sample; and providing instructions to a user to allow the user to use the kit to identify the presence of at least two analytes in the fluid sample.
- Thus, in accordance with the presently disclosed and claimed inventive concept(s), there have been provided devices, methods, and kits that allow a small volume of a single fluid sample obtained from a patient to be re-used in order to detect at least two analytes present in such single fluid sample. Accordingly, a multiplexed panel associated with the various analytes present in the single fluid sample is created that fully satisfy the objectives and advantages set forth hereinabove. Although the presently disclosed and claimed inventive concept(s) has been described in conjunction with the specific drawings, experimentation, results and language set forth hereinabove, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the presently disclosed and claimed inventive concept(s).
Claims (18)
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