Western blotting is a very useful and common laboratory procedure. In the typical procedure, protein mixtures are separated on polyacrylamide gel into bands using an applied electric field. After the proteins are separated into bands, the separated bands are transferred to a membrane. After transfer, the separated proteins are probed with primary and secondary antibodies for detection. The detection can be accomplished via radioactivity, chemiluminescence, fluorescence, or absorbance. The most common detection method is chemiluminescence; however, in order to detect multiple analytes simultaneously, fluorescence has recently gained popularity.
Western blotting is time consuming and laborious due to the number of steps involved in the process. Western blotting requires gel electrophoresis to separate the proteins. The proteins are then transferred to a membrane (e.g., nitrocellulose), where they are normally identified with a primary antibody and detected with a secondary antibody.
More recently, other Western blot techniques have been developed (W. Pan et al., Anal. Chem. 2010, 82, 3974-3976). For example, a microfluidic Western blot has been developed that incorporates molecular weight markers and has good capacity to analyze multiple proteins simultaneously. However, this technique requires transfer from a polyacrylamide gel to a polyvinylidene fluoride (PVDF) membrane.
In view of the foregoing, what is needed in the art are new systems and methods for separating mixtures of biologically interesting molecules. The present invention satisfies these and other needs.
The present invention provides systems and methods which enable or utilize an immuno-based assay, such as a Western immunoassay, to separate, detect or to monitor an analyte such as a biomolecule.
As such, in one embodiment, the present invention provides an electrophoresis system useful for a Western immunoassay, the electrophoresis system comprising:
In another embodiment, the present invention provides a Western immunoassay method, the method comprising:
In yet another embodiment, the present invention provides a kit, the kit comprising:
These and other aspects, objects and advantages will become more apparent when read with the detailed description and figures which follow.
As used herein, certain terms may have the following defined meanings. As used in the specification and claims, the singular form “a,” “an” and “the” include singular and plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a single cell as well as a plurality of cells, including mixtures thereof.
The term “about,” as used to modify a numerical value, indicates a defined range around that value. If “X” were the value, “about X” would generally indicate a value from 0.95X to 1.05X. Any reference to “about X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, “about X” is intended to teach and provide written description support for a claim limitation of, e.g., “0.98X.” When the quantity “X” only includes whole-integer values (e.g., “X carbons”), “about X” indicates from (X−1) to (X+1). In this case, “about X” as used herein specifically indicates at least the values X, X−1, and X+1.
A “silica colloidal crystal” includes a plurality of silica particles packed in a repeating pattern in two or three dimensions. The crystal can be monocrystalline (containing a single unit cell having one periodic arrangement) or polycrystalline (including two or more unit cells having the same or different periodic arrangements, forming a plurality of crystal grains). The arrangement of the silica particles in the unit cell is analogous to the arrangement of atoms or molecules in a conventional crystal. The silica colloidal crystal contains space (i.e., interstitial space) between individual particles.
“Introducing” a sample into a separation bed can include filling (or partially filling) the interstitial spaces between particles with the sample, or as otherwise known in the art. Introducing the sample can include injection of the sample via pressure, gravity, or electrostatic force.
An “array” refers to a grouping or an arrangement, without necessarily being a regular arrangement.
The terms “antibody,” “immunoglobulin”, “Ab,”, “Ig,” “anti-target,” and like terms include a polypeptide encoded by an immunoglobulin gene or functional fragments thereof that specifically binds and recognizes an antigen. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. The term antibody activity, or antibody function, refers to specific binding of the antibody to the antibody target.
An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms “variable heavy chain,” “VH,” or “VH” refer to the variable region of an immunoglobulin heavy chain, including an Fv, scFv, dsFv or Fab; while the terms “variable light chain,” “VL” or “VL” refer to the variable region of an immunoglobulin light chain, including of an Fv, scFv, dsFv or Fab. The C-terminus of the heavy chains forms the constant region, often referred to as the “Fc” region.
Examples of antibody fragments that bind antigens include, but are not limited to, complete antibody molecules, antibody fragments, such as Fv, single chain Fv (scFv), complementarity determining regions (CDRs), VL (light chain variable region), VH (heavy chain variable region), Fab, F(ab′)2 and any combination of those or any other functional portion of an immunoglobulin peptide capable of binding to target antigens (see, e.g., F
The term “polyclonal antibody” includes a heterogeneous population of antibodies raised against an antigen. The antibodies within the population usually bind to different epitopes on the antigen. Polyclonal antibodies can be useful for detecting an antigen in a broader range of conditions than monoclonal antibodies, which are specific for a particular epitope.
The term “monoclonal antibody” includes a clonal population of antibodies that bind to the same epitope on an antigen. Monoclonal antibodies can be made by selecting a single member of a heterogeneous population of antibodies for antigen binding, and clonally expanding the cell that produces that antibody in a hybridoma cell (see, e.g., Kohler and Milstein (1975) Nature 256:495). Recombinant methods are also used for preparing monoclonal antibodies (see, e.g., Chadd and Chamow (2001) Curr. Opin. Biotechnol. 12:188094).
The phrase “specifically (or significantly or selectively) binds to” when referring to a given target, includes a binding reaction which is determinative of the presence of the target in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, a target-specific antibody will bind to the target and not bind in a significant amount to other proteins and molecules present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular target. For example, antibodies raised against a selected target can be further selected to obtain antibodies that do not bind to other molecules.
One of skill will understand that “specific” or “significant” binding are not intended to be absolute terms. For example, if an antibody does not significantly bind to a particular epitope, it binds with at least 5-fold, 8-fold, 10-fold, 20-fold, 50-fold, 80-fold, or 100-fold reduced affinity as compared to the epitope against which the antibody was raised. For example, a target-specific antibody does not significantly bind to a non-target if it binds to the latter with less than 20%, 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less affinity than to the target. Binding affinity can be determined using techniques known in the art, e.g., ELISAs. Affinity can be expressed as dissociation constant (Kd or KD). A relatively higher Kd indicates lower affinity. Thus, for example, the Kd of a target-specific antibody for the target will typically be lower by a factor of at least 5, 8, 10, 15, 20, 50, 100, 500, 1000, or more than the Kd of the target-specific antibody with a different molecule. One of skill will understand how to design controls to indicate non-specific binding and compare relative binding levels.
The term “primary antibody” will be understood by one of skill to refer to an antibody or fragment thereof that specifically binds to an analyte (e.g., substance, antigen, component) of interest. The primary antibody can further comprise a tag, e.g., for recognition by a secondary antibody or associated binding protein (e.g., GFP, biotin, or streptavidin). A binding moeity such as an aptamer or affibody can also be used.
The term “secondary antibody” refers to an antibody that specifically binds to a primary antibody. A secondary antibody can be specific for the primary antibody (e.g., specific for primary antibodies derived from a particular species) or a tag on the primary antibody (e.g., GFP, biotin, or streptavidin). A secondary antibody can be bispecific, e.g., with one variable region specific for a primary antibody, and a second variable region specific for a bridge antigen. A binding moeity such as an aptamer or affibody can also be used.
A “biological marker” is a biomolecule, a biochemical label, or other biological motif that identifies a structure or function of interest in a biological specimen/sample.
“Biological assay” is a method of biological analysis, in which a biological substrate of interest is reacted with chemicals or biochemicals, where the reaction can be used to characterize the substrate (e.g., by function, presence or absence, etc.). Examples of biological assays are innumerable, and include DNA sequencing, restriction fragment length polymorphism determination, Southern blotting and other forms of DNA hybridization analysis, determination of single-strand conformational polymorphisms, comparative genomic hybridization, mobility-shift DNA binding assays, protein gel electrophoresis, Northern blotting and other forms of RNA hybridization analysis, protein purification, chromatography, immunoprecipitation, protein sequence determination, Western blotting (protein immunoblotting), ELISA and other forms of antibody-based protein detection, isolation of biomolecules for use as antigens to produce antibodies, PCR, RT PCR, differential display of mRNA by PCR, and the like. Protocols for carrying out these and other forms of assays are readily available to those skilled in the art.
“Detecting” refers to determining the presence, absence, or amount of an analyte in a sample, or as otherwise known in the art. It can include quantifying the amount of the analyte in a sample or per cell in a sample.
A “biomolecule” includes a molecule of a type typically found in a biological system, whether such molecule is naturally occurring or the result of some external disturbance of the system (e.g., a disease, poisoning, genetic manipulation, etc.), as well as synthetic analogs and derivatives thereof. Non-limiting examples of biomolecules include amino acids (naturally occurring or synthetic), peptides, polypeptides, proteins, glycosylated and unglycosylated proteins (e.g., polyclonal and monoclonal antibodies, receptors, interferons, enzymes, etc.), nucleosides, nucleotides, oligonucleotides (e.g., DNA, RNA, PNA oligos), polynucleotides (e.g., DNA, cDNA, RNA, etc.), carbohydrates, hormones, haptens, steroids, and toxins, etc. Biomolecules may be isolated from natural sources, or they may be synthetic.
The term “equilibrium dissociation constant” or “affinity” abbreviated (Kd or KD), refers to the dissociation rate constant (kd, time−1) divided by the association rate constant (ka, time−1 M−1). Equilibrium dissociation constants can be measured using any known method in the art. As an example, the affinity for Protein A/G for Ig molecules is typically in the low nM range. Antibodies with high affinity for an antibody target have a monovalent affinity less than about 100 nM, and often less than about 50 nM, 1 nM, 500 pM or about 50 pM as determined by surface plasmon resonance analysis performed at 37° C.
A “specific binding agent” is an agent that recognizes and binds substantially preferentially to a biological marker of interest, so that the agent provides potentially useful information about the biological marker. Examples of specific binding agents are polyclonal and monoclonal antibodies for an antigen of interest; proteins and protein derivatives that interact or bind to receptors (for example, calmodulin or a labeled calmodulin derivative) and nucleic acid probes such as DNA and RNA probes.
The term “sample,” e.g., with reference to a detection assay, is used broadly. The term can refer to a sample at any stage, e.g., a crude biological sample, a sample of pre-selected cells, a component of a biological sample, a sample deposited on a substrate, or separated electrophoretically or using chromatography. Typical examples of a sample includes cell lysate, a solution of proteins or other biomolecules, a population of cells, a biopsy, biological fluid (e.g., blood, blood component, mucus, urine, lymph, saliva, tears, etc.), or a tissue section. A “sample” can be any mixture or pure substance having at least one analyte, or a sample as otherwise known in the art. An “analyte” includes a substance of interest such as a biomolecule. Biomolecules are molecules of a type typically found in a biological system, whether such molecule is naturally occurring or the result of some external disturbance of the system (e.g., a disease, poisoning, genetic manipulation, etc.), as well as synthetic analogs and derivatives thereof. Non-limiting examples of biomolecules include amino acids (naturally occurring or synthetic), peptides, polypeptides, glycosylated and unglycosylated proteins (e.g., polyclonal and monoclonal antibodies, receptors, interferons, enzymes, etc.), nucleosides, nucleotides, oligonucleotides (e.g., DNA, RNA, PNA oligos), polynucleotides (e.g., DNA, cDNA, RNA, etc.), carbohydrates, hormones, haptens, steroids, toxins, etc. Biomolecules can be isolated from natural sources, or they can be synthetic.
A “control” refers to a value that serves as a reference, usually a known reference, for comparison to a test reagent, assay, sample or set of conditions. For example, a test assay can include a sample exposed to a test condition or a test agent, while the control is not exposed to the test condition or agent (e.g., a primary antibody). The control can also be a positive control, e.g., a known assay exposed to known conditions or agents, for the sake of comparison to the test condition (e.g., a standard two-step immunoassay using a secondary antibody). A control can also represent an average value gathered from a plurality of samples, e.g., to obtain an average value. A control value can also be obtained from the same sample or batch of reagent, e.g., from an earlier-obtained sample or batch prior to storage. One of skill will recognize that controls can be designed for assessment of any number of parameters.
One of skill in the art will understand which controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant.
The present invention provides systems and methods which enable or utilize an immunologically-based assay, such as a Western immunoassay, to separate, detect or to monitor an analyte such as a biomolecule. The preferred method of the invention utilizes a sensitive Western immunoassay to separate and detect proteins. Advantageously, the systems, methods and kits can be used in sensitive diagnostic methods.
A. Systems and Methods
The present invention provides systems and methods for separating molecules such as biomolecules. In one embodiment, the present invention provides an electrophoresis system useful for a Western immunoassay, the electrophoresis system comprising:
In certain aspects, the electrophoresis systems and methods of the present invention include nanoparticles 117 such as individual particles, wherein the nanoparticles are made of silica. In certain aspects, the silica nanoparticles are arranged in a regular, crystalline structure. In certain aspects, the crystal structure is body centered cubic. Each of the plurality of nanoparticles is between about 1 nm to about 2000 nm in diameter, more specifically between about 1 nm to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, or 2000 nm in diameter. The population of nanoparticles can be monodisperse or polydisperse. Larger particles, with diameters on the order of a few microns (2, 3 or 4 μm), can also be used in the methods of the invention. In certain aspects, the colloidal silica nanoparticles are spheres having a diameter of about 1 μm, thereby resulting in a minimum interstitial space size (or pores) of about 155 nm and a surface-to-volume ratio of about 13. The pore size can range from 0.15 nm to 309 nm or more (see, Example 9).
In certain aspects, the present invention provides a pluality of colloidal nanoparticles which comprise substantially monodisperse colloidal nanoparticles, which gives rise to a monodisperse interstitial pore size. A monodisperse pore size increases the separation efficiency, giving increased separation resolution when samples are separated over the same distance. An increase in the separation efficiency leads to shorter separation lengths, increased resolution, or decreased separation times. In certain aspects, the colloidal nanoparticles are about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100% monodisperse.
In one aspect, the power for applying a voltage along the substrate between terminals 120 and 125 supplies an electric field having voltages of about 1 V cm−1 to 2000 V cm−1, such as about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, or about 2000V cm−1. Higher voltages or varied voltages can also be used, depending on the particular separation methods employed.
In certain preferred aspects, the electrophoresis system of the present invention is used in performing a Western immunoassay and includes a means for applying a detection reagent. As is illustrated in
As shown in
Turning now to
A cut-a-way side view is shown in
Each individual well can be loaded with samples 301 (now darkened) as shown in
As shown in
As shown in
In an alternative embodiment as shown in
In one aspect, each of the nanoparticles with the separation bed is less than 2 μm (e.g., 1 μM or 1.5 μm) in diameter, and together they form a stationary phase for analytical separation. The nanoparticles are each preferably nonporous. In certain instances, larger nanoparticle sizes can result in a decrease in the ratio of surface to volume and an increase in the pore radius. This can decrease the selectivity and increase the migration speed of the analyte. In contrast, the smaller the nanoparticle size, the surface area to volume ratio increases and the pore radius decreases. This typically results in better or increased resolution.
In certain aspects, the population of nanoparticles are uncoated, coated, or a mixture of uncoated and coated. In certain aspects, the nanoparticles may be coated with either non-covalent coatings, covalent coatings or a combination thereof. In one aspect, a protein-free blocking buffer is used as a non-covalent coating for the nanoparticles (e.g., Pierce 37584 Protein-Free (PBS) Blocking Buffer). In certain aspects, buffers that supress or elimiate eletroosmotic flow (EOF) are used with uncoated nanoparticles (e.g., formic acid containing buffers).
In certain aspects, the nanoparticles are coated, such as with a polymer coat. The polymer modification can be a hydrophobic or a hydrophilic polymer. The nanoparticles can be a mixture of different types of coatings. Suitable hydrophilic polymers include, but are not limited to, polyalcohols, polyoxyethylenes, polyethers, polyamides, polyimides, polycarboxylates, polysulfates, polysufonates, polyphosphates, polyphosphonates and a combination thereof. In certain aspects, the polyamide hydrophilic polymer is a polyacrylamide.
In certain aspects, the polymer forms a brush layer on the plurality of nanoparticles. In one instance, the brush layer is a polyacrylamide. The bed of coated nanoparticles forms a matrix of silica particles ideal for separation.
In certain aspects, the hydrophilic polymer layer is further functionalized for immobilization of an analyte. In one aspect, the immobilization of the analyte is covalent. Alternatively, the immobilization is noncovalent. In certain aspects, the functionalization for immobilization of an analyte is effectuated by UV light, by a change in pH, or by precipitation. For example, proteins can be precipitated or fixed via an acid solution. Alternatively, proteins can be immobilized on separation bed via photoactivation of a benzophenone moiety which generates a highly reactive triplet intermediate to form a covalent bond. Further, a radical species can react with tyramide to amplify signals.
In another embodiment, the present invention provides an electrophoretic method, the method comprising:
In certain embodiments, a colloidal crystal nanoparticle, such as a plurality of silica colloidal crystals are disposed on a surface or substrate of a device. In one aspect, a sample, which can be a mixture of proteins or biological molecules, is placed into the device and an electric field between terminals is applied. In one instance, the mixture of analytes to be separated is placed at the cathode end. In another aspect, the mixture of analytes to be separated is placed at the anode end. In certain other instances, the sample can be placed anywhere between the cathode and anode.
In certain aspects, a sample for analysis is loaded on the inventive device and the power supply is used to electrophoretically separate the sample. The electrophoresis system can then separate the mixture in a second dimension based on the charge and or size of each of the molecules in the mixture. The separation bed is used to separate proteins as a function of size, pI, or other useful characteristics of the analyte using electrophoresis, pressure-driven flow, convection, or other flow.
After the mixture is separated (either in 1-dimension, 2-dimensions or multiple dimensions), the analytes (such as proteins) are immobilized on the nanoparticles using for example, pH, light or precipitation. Advantageously, there is no need to transfer or blot the separated molecules to a membrane.
The colloidal crystal with immobilized molecules (e.g., proteins) is optionally washed and then contacted, incubated or submerged in an antibody solution (blocking buffer may or may not be necessary depending on the bead coating) or with antibodies by a means for applying a detection reagent. The antibodies are specific for the analyte to be detected. In certain instances, this antibody is the “first antibody.” The substrate is then washed with a low salt buffer to remove non-binding antibodies and proteins.
The washed substrate is then incubated with a “second antibody” or antibodies that recognize the first antibody. In certain instances, the second antibody has a reporter group attached for the visualization of the first antibody. The reporter that is attached to the second antibody can be either a chemical “tag” or an enzyme (such as alkaline phosphatase, horseradish peroxidase) that catalyzes a reaction that converts an added substrate to a product that is visualized.
In some aspects, the tag is a label such as a fluorophore. In some embodiments, the label is a cyanine dye, e.g., a near IR dye (e.g., IRDye® 680RD, IRDye® 800CW or IRDye® 680LT). In some aspects, the primary antibody and/or secondary antibody can be a mammalian IgG, e.g., from a human, mouse, rat, goat, rabbit, horse, guinea pig, sheep, hamster, swine, bovine, cat, dog, or monkey. In some embodiments, the antibody is selected from the group consisting of human IgG, human IgA, human IgE, human IgM, and human IgD. In some embodiments, the antibody is a polyclonal antibody.
In one aspect, the nanoparticles are coated with a non-specific protein immobilization moiety that is activated post-separation (e.g. UV-light), or electrolytes are introduced to initiate a pH-dependent reaction by electrophoresis or pressure, or the colloidal crystal is submerged in an electrolyte.
In one aspect, the colloidal crystal can enable multiple ‘lanes’ either as a single, continuous entity in which diffusion does not allow cross-reactivity between lanes, or the colloidal crystal can be printed as individual lanes with a barrier between (if necessary). After washing, the antigen and ladder may be detected in the separation bed by various means such as an optical means (e.g., fluorescence).
In certain aspects, a colloidal crystal structure is a densely ordered packing of particles in a patterned arrangement (e.g. face-centered cubic) with long range order that is generally hundreds of particles long, but may be more or less particles in length, and typically extends in two or three dimensions. A coloidal crystal is most often composed of spherically shaped particles, but may be composed of non-spherical particles as well (Langmuir 2007, 23, 8810-8814). A colloidal crystal may be composed of monodisperse particles or may also be composed of multiple particle sizes in an arrangement such that a predictable pattern is created. One of the most common techniques used to create colloidal crystals is Evaporative Induced Self-Assembly (EISA).
In certain aspects, the smaller the particle size the better separation of small proteins. For example, proteins of molecular weight 5-30 kDa are separated with nanoparticles having a diameter of 100 nm to 500 nm. In other aspects, proteins of molecular weight 30-100 kDa are separated with nanoparticles having a diameter of 500 nm to 700 nm. In still other aspects, proteins of molecular weight 100-500 kDa are separated with nanoparticles having a diameter of 700 nm to 1000 nm.
The method of visualization can include chemiluminescent, chemifluorescence, fluorescence, or colorimetric technologies. In one instance, a visualization method is chemiluminescent and based on a peroxidase (e.g., horseradish peroxidase) reporter conjugated to the second antibody or a fluorescent label.
In another aspect, the present invention provides for multiple dimension (such as 2-D or 3-D or more) separation. For example, in one embodiment, isoelectric focusing can be used in a first dimension, followed by a separation along a second dimension. Suitable second dimension separation techniques include, but are not limited to, capillary electrophoresis, thin layer chromatography, high pressure chromatography, size exclusion chromatography and the like.
In certain other aspects, the present invention provides various lanes for the analytes to be separated in one-dimension. The space between the lanes may either be a solid (e.g. part of the removable lid), or the space may be empty (i.e., dry, gaseous). The colloid crystals provide a strong capillary force, therefore, if one pipetted samples onto the individual lanes (of certain volumes) the sample remains within a given lane. If the separation method is IEF, the samples do not have to be ‘stacked’ at either end, therefore, the lane configuration is convenient for keeping samples separate and distinct, while allowing 1-D separation.
In certain aspects, the electrophoresis system of the present invention has a substrate comprising an x-axis and a y-axis and a voltage is configured to resolve or separate an analyte along the x-axis as a first dimension. In one aspect, the system is configured to separate or resolve an analyte along the y-axis as a second dimension. In one aspect, the resolution or separation is performed using the size of the analyte. Separations in multiple dimensions are possible.
In certain aspects, the electrophoresis systems and methods of the present invention resolve or separate the analyte as a function of the pI of the analyte. The isoelectric point (pI) is the pH at which a particular molecule carries no net electrical charge. Other suitable techniques for resolution or separation include, but are not limited to, electrophoresis, isoelectric focusing, and chromatography.
Turning now to
In one aspect, as shown in
In
In
Suitable second dimension separation techniques include, but are not limited to, capillary electrophoresis, thin layer chromatography, high pressure chromatography, size exclusion chromatography and the like.
Resolved analytes can also be visualized in the separation bed. In certain aspects, proteins can be labeled with a dye (such as a fluorescent dye) before or after separation. Examples of suitable dyes include, but are not limited to, rhodamines, fluoresceins, coumarins, and cyanines. In certain aspects, a cyanine dye can be a near IR dye (e.g., IRDye® 680RD, IRDye® 800CW or IRDye® 680LT). Alternatively, resolved analytes can be detected using antibodies or combinations of antibodies that are specific for analytes of interest. In some aspects, a primary antibody and/or a secondary antibody can be a mammalian IgG, e.g., from a human, mouse, rat, goat, rabbit, horse, guinea pig, sheep, hamster, swine, bovine, cat, dog, or monkey. The antibody can be, for example, human IgG, human IgA, human IgE, human IgM, and human IgD. The antibody can be a monoclonal antibody or a polyclonal antibody. Labeled analytes and labeled analyte/antibody conjugates can be detected, for example, with a fluorescence scanner. Depending on the detection strategy, visualization of resolved analytes can also include chemiluminescent and colorimetric methods as is known in the art. For example, resolved analytes can be visualized using a horseradish peroxidase (HRP) reporter conjugated to an antibody and subjecting the enzyme with a substrate to produce a product with an emission of light. Chemiluminescent substrates for horseradish peroxidase (HRP) are typically two-component systems consisting of a peroxide solution and an enhanced luminol solution. After mixing the two components together and incubating with HRP-conjugated antibodies, a chemical reaction produces light.
B. Kits
The present invention further provides kits for convenient immunoassays such as Western immunoassays. The kit contains a substrate with nanoparticles useful for carrying-out a Western immunoassay. In some aspects, the kit comprises tubes for detection reagents. In some aspects, the detection reagents are lyophilized, and a container with solution for reconstitution is provided in the kit. In other embodiments, the kit includes wash or blocking buffers compatible with the substrate. In still other embodiments, the kits contain protein ladders or mixtures of highly purified proteins, which resolve into clearly identifiable sharp bands. The sizes can range from 5-500 kDa or more. The protein ladder is intended for use as a precise size standard when performing electrophoresis to calculate the molecular weight of a protein of interest.
Kits according to the invention typically include instructions for use. For example, in a simple embodiment of the antibody labeling solution, the instructions can recite the protocol for adding a desired antibody to the analyte to be detected.
The diagnostic kits and immunoassays described herein can be used to diagnosis diseases or disorders such as, but not limited to, Lyme disease, other tick-borne disease, Creutzfeldt-Jakob Disease, prion disease, HIV infection, HSV infection, HCMV infection, SARS infection, Helicobacter pylori infection, Campylobacter pylori infection, Parvovirus infection, Hepatitis C infection, Kaposi's sarcoma virus infection, influenza infection, other viral infections, bacterial infection, Staphylococcus aureus infection, fungal infection, paraneuplastic syndrome, amyotophic lateral sclerosis (ALS), spinal muscular atrophies (SMA), primary lateral sclerosis (PLS), Arthrogryposis Multiplex Congenita (AMC), Alzheimer's disease, heart failure severity, lung cancer, pancreatic cancer, colorectal cancer, prostate cancer, bladder cancer, gastric cancer, oral cancer, breast cancer, ovarian cancer, lymphoma, metastasis, neoplasia, COPD, kidney disase, Sjogren's syndrome, autism, depression, neuropsychiatric disease, inflammatory disease, autoimmune disease, myasthenia gravis, scleroderma, osteoporosis, and the like. Detailed descriptions of diagnostic method based on western blotting are found in, e.g., U.S. Pat. Nos. 8,962,257; 8,145,112; 8,257,917; 7,709,208; 7,192,698; 6,013,460; and 5,545,534.
Controls can also be included, e.g., containers with labeled proteins and known antibody and antibody targets to run alongside the desired sample. Controls can also include known antibodies and/or antibody targets at preset concentrations, e.g., for titering a desired assay.
A kit can also include a plurality of containers with labeled antibodies such as with different labels that are detected in different conditions (e.g., different fluorophores or enzymes. The plurality of containers can also include antibodies with different amounts of covalently attached label, e.g., for detection of relatively abundant vs. rare targets.
A kit according to the invention can include a panel of known antibodies, e.g., for detection of certain conditions. For example, a colorectal cancer-specific detection kit can include antibodies to known CRC markers. The kit can also include positive and negative target controls.
In some embodiments, the kit includes assay components, e.g., for running an ELISA, or Western immunoassay. Thus, for example, a Western immunoassay kit can include solutions and reagents for carrying out the immunoassay. In some embodiments, the Western immunoassay kit can include containers with antibodies, optionally with positive and negative control samples. In some embodiments, the user will provide the desired test sample and the target-specific antibody to be labeled according to the invention.
In some embodiments, chemiluminescent reagents can be used. For Western blot detection methods, chemiluminescence is a preferred the detection method. Suitable reagents include luminol-based chemiluminescent substrates. Chemiluminescent substrates for horseradish peroxidase (HRP) are two-component systems consisting of a stable peroxide solution and an enhanced luminol solution. To make a working solution, the two components are mixed together and incubated with a blot on which HRP-conjugated antibodies (or other probes) are bound, in the presence of HRP, a chemical reaction produces light that can be detected by film or detector.
In some embodiments, the kit also includes components for immunoprecipitation, e.g., beads or other matrix associated with a capture agent. For example, the matrix can be coated with a substance that has affinity for antibodies or for a particular tag (e.g., Ni resin, strepavidin, etc.), or for proteins or nucleotides generally.
Silica nanoparticles having an approximate diameter of 250 nm, 500 nm, or 750 nm were purchased from Fiber Optic Center, Inc. (New Bedford, Mass.), and were then calcined at 600° C. for 12 h. Glass or quartz microscope slides were purchased. The silica nanoparticles were deposited onto the glass microscope slides using a draw-down coater, forming a highly-ordered three-dimensional silica colloidal crystal. The nanoparticles were then coated with a brushed layer of polyacrylamide.
A Western immunoassay is compared to that of a traditional two-step detection using IRDye® 680LT labeled secondary antibodies.
In a traditional Western blot, serial dilutions of rabbit IgG are added to a C32 cell lysate. The lysate components are separated by SDS-PAGE and immobilized on membranes. The blots are probed with (1) mouse anti-rabbit IgG primary antibody followed by (2) IRDye® 680LT labeled goat anti-mouse secondary antibody (Control).
In the Western immunoassay, a C32 cell lysate is separated on a silica colloidal crystal and immobilized by precipitation. The crystals are probed with (1) mouse anti-rabbit IgG primary antibody followed by (2) IRDye® 680LT labeled goat anti-mouse secondary antibody (test sample).
The results indicate that the limit of detection (LOD) for the in-crystal Western immunoassay is comparable to a traditional Western blot.
A glass slide is chemically modified with a solution of n-butyldimethylchlorosilane in anhydrous toluene under nitrogen. The slide is then rinsed with dry toluene and dried under vacuum at 80° C. A 1-mm-wide stripe of 1 cm in length is masked off on the slide and chemically etched with an ammonium bifluoride salt paste. A second, chemically modified glass slide is used to cover the channel, and the assembly is secured using binder clips. A 10% w/w silica colloid is wicked into the channel and allowed to dry at room temperature. After drying, the cover glass slide is removed and the packed channel is silylated with a polymerization initiator. Linear polyacrylamide chains are grown using a complex of CuCl with tris(2-dimethylaminoethyl) amine as the catalyst; the slide is immersed in a solution of acrylamide monomer and CuCl catalyst and the mixture is allowed to polymerize.
The slide with the packed channel is wetted with running buffer (25 mM Tris; 192 mM glycine; 0.1% (w/v) SDS; pH 8.0) and covered with a PDMS seal at each end of the channel to prevent drying. The channel is electrically conditioned at 50 Vcm−1 using a high-voltage power supply until the current becomes static.
Proteins (rabbit IgG, myoglobin from equine skeletal muscle, cytochrome c from bovine heart and lysozyme from chicken egg white) are dissolved in PBS buffer and combined in a denaturation buffer (62 mM Tris; 1 mM EDTA; 3% sucrose; 2% SDS; pH 8.0). The concentration of each protein is about 0.05 mg/mL. The proteins are denatured at 100° C. for 3 minutes.
Proteins are electrokinetically loaded into the prepared channel under 300 Vcm−1 for 30 s. Next, the channel is mounted between buffer-filled reservoirs and an electric field of 50 Vcm−1 is used for separation. Following separation, the channel is briefly rinsed in deionized water, and the resolved proteins are fixed in the polyacrylamide brush layer using a mixture of methanol, deionized water, and acetic acid (50:45:5, v:v:v). The crystals are probed with antibodies to detect the resolved proteins. The rabbit IgG is detected by probing with (1) mouse anti-rabbit IgG primary antibody, followed by (2) IRDye® 680LT labeled goat anti-mouse secondary antibody. The myoglobin is detected by probing with (1) mouse anti-myoglobin IgG primary antibody, followed by (2) IRDye® 800CW labeled goat anti-mouse secondary antibody. 0.1% Tween-20 is included in the primary antibody solution. The crystal is briefly washed with PBS containing 0.1% Tween-20 before applying the secondary antibody solution. The secondary antibody solution contains 5% w/v non-fat milk. The crystal is washed briefly with PBS before imaging using an IR scanner.
A pH gradient is established across a channel packed with a silica colloidal crystal (fabricated as described above) using a commercially available carrier ampholyte mixture
Proteins (rabbit IgG, myoglobin from equine skeletal muscle, cytochrome c from bovine heart and lysozyme from chicken egg white) are dissolved in PBS and combined in an isoelectric focusing solution (8 M urea; 20 mM DTT; 0.5% Triton X-100). The concentration of each protein is about 0.05 mg/mL.
Proteins are electrokinetically loaded into the prepared channel under 300 Vcm−1 for 30 s. Isoelectric focusing is conducted by ramping the voltage from 50 Vcm−1 to 1000 Vcm−1 over a period of time sufficient for separation of the proteins.
The channel is aligned with a second channel equilibrated with SDS running buffer (25 mM Tris; 192 mM glycine; 0.1% (w/v) SDS; pH 8.0). A voltage of 50 Vcm−1 is applied across the aligned channels to separate the proteins according to size. Following separation, the channel is briefly rinsed in deionized water, and the resolved proteins are fixed in the polyacrylamide brush layer using a mixture of methanol, deionized water, and acetic acid (50:45:5, v:v:v). The crystals are probed with antibodies to detect the resolved proteins. The rabbit IgG is detected by probing with (1) mouse anti-rabbit IgG primary antibody, followed by (2) IRDye® 680LT labeled goat anti-mouse secondary antibody. The myoglobin is detected by probing with (1) mouse anti-myoglobin IgG primary antibody, followed by (2) IRDye® 800CW labeled goat anti-mouse secondary antibody. 0.1% Tween-20 is included in the primary antibody solution. The crystal is briefly washed with PBS containing 0.1% Tween-20 before applying the secondary antibody solution. The secondary antibody solution contains 5% w/v non-fat milk. The crystal is washed briefly with PBS before imaging using an IR scanner.
An automatic drawdown machine was used to coat a clean glass slide with an approximately 30% silica colloid slurry in ethanol. The silica slurry was allowed to dry on the glass slide. Silica coated glass slides were placed face up in a covered glass Petri dish in a fume hood. The glass slides were flushed with Argon for several minutes. Using a syringe and needle, a total of 100 μL silicon tetrachloride (SiCl4) was placed on the bottom of the dish in several locations and the dish was flushed with argon for one minute. Vapor deposition of the SiCl4 was allowed to take place for 5 minutes. After the vapor deposition was complete, the process was repeated for additional time to ensure sufficient deposition of the SiCl4.
Silica coated slides were placed in a beaker with 1M nitric ccid. The 1M nitric acid was heated to reflux for 1 hour. The 1M nitric acid was decanted and the slides were washed with Milli-Q water followed by an ethanol wash. The slides were dried at 60° C. for 24 hours. Re-hydroxylated silica coated slides were washed with toluene followed by a 30 minute exposure to a solution of 2% ((chloromethyl)phenyl)trichlorosilane and 0.1% methyltrichlorosilane in toluene. The slides were then washed with toluene and dried at 120° C. for 3 hours.
Re-hydroxylated and silanized silica coated slides were washed with water: 2-propanol (1:1) followed by a 3 hour exposure (under argon gas) to a solution of 0.5M acrylamide, 10 mM copper chloride, 10 mM Tris[2-(dimethylamino)ethyl]amine and 8 mM L-ascorbate in water: 2-propanol (1:1). The slides were washed with water: 2-propanol (1:1) and then dried at 120° C. for 3 hours.
To test protein binding on the silica coated slide surface with the addition of a protein precipitation solution, 1 μl of a 10 ng/μl IRDye® 680LT Goat anti-mouse IgG solution was spotted onto the silica coated slide. The slide was imaged using a LI-COR Odyssey® Imaging System and the image was recorded. The slide was then either incubated in 40% methanol with 10% trichloroacetic acid (TCA) for 1 hour, or left untreated. The treated slides were rescanned on the Odyssey® Imaging-System. The slides were then placed in Odyssey® Blocking Buffer for 1 hour, washed with a PBS solution and rescanned on the Odyssey® Imaging System. The slides were then incubated for 1 hour in a IRDye® 800CW Donkey anti-goat IgG solution. The slides were washed for 30 minutes in PBS and the 800 nm channel was imaged on the Odyssey® Imaging System to identify binding of the IRDye® 800CW Donkey anti-goat IgG.
A 1.8-cm long channel was packed with silica particles that were coated with a polyacrylamide brush layer. The ends of the channel were fitted with reservoirs that could be used for loading the channel and for electrophoresis. A platinum electrode was inserted into each reservoir and connected to a power supply. The channel was imaged by placing the chip on an inverted fluorescence microscope, and using fluorescence-labeled proteins in the experiments.
4% Carrier ampholytes pH 3-10, 8 M urea, 2% CHAPS and 50 mM dithiothreitol was applied to the whole channel, along with a mixture of carbonic anhydrase I and II and lectin glycoprotein, all of which were labeled with Alexa Fluor 546. The catholyte reservoir was filled with 20 mM NaOH and the analyte reservoir was filled with 20 mM H3PO4. The electric field was supplied by a high-voltage power supply to produce approximately 100V/cm. The proteins were allowed to migrate to their isoelectric points and the channel was imaged with an inverted optical microscope equipped with an excitation source and filter set suitable to image the 546 nm labeled proteins. The channel was opened so that one surface of the entire length of the channel was accessible.
Silica particles were calcined at 600° C. three times for 6 h each in a box furnace, then rehydroxylated in 50% nitric acid, and finally suspended in ultrapure water and used to make a 30% (w/w) slurry. Fused silica capillaries of 100 μm i.d. with Teflon coatings were cleaned by pumping 0.1 M NaOH for 15 min, then rinsed with ultrapure water and ethanol for 20 min each, and then cut into 12 cm sections and dried in a vacuum oven at 70° for 30 min.
Silica slurries were prepared at a 30% w/w concentration in water and sonicated in a water bath. Slurries were wicked into capillaries of 12 cm in length, and then packed under pressure at 345 bar with sonication for 15 min using a 0.5 μm frit. After packing, the fit was removed and the capillary was allowed to dry in a dessicator.
PDMS buffer reservoirs were placed on a glass slide to prevent electrophoresis gas bubbles from entering the channel. The packed capillaries packed were saturated with a buffer containing H2O with 0.1% Formic acid and 0.1% SDS overnight. Protein samples labeled with a 546 nm fluorescent probe were and loaded into the capillaries by diffusion. The capillary was then mounted between the dual PDMS reservoirs in a tight-fitting notch, with the protein loaded at the cathode end. Platinum electrodes were placed in the outer reservoir wells. The reservoirs were then filled with sample running buffer, and an electric field of 100 Vcm-1 was applied. The total separation length of the packed capillary was approximately 3.8 cm.
The proteins were allowed to migrate through the packed capillary and separate according to size. The labeled proteins were imaged at one end of the capillary with an inverted optical microscope equipped with an excitation source and filter set suitable to image the 546 nm labeled proteins (see,
Silica nanoparticles having an approximate diameter of 250 nm, 500 nm, or 750 nm were purchased from Fiber Optic Center, Inc. (New Bedford, Mass.), and were then calcined at 600° C. for 12 hours. Glass or quartz microscope slides were purchased. The silica nanoparticles were deposited onto the glass microscope slides using a draw-down coater, forming a highly-ordered three-dimensional silica colloidal crystal. The nanoparticles were then coated with a brushed layer of polyacrylamide.
The nanoparticles themselves are mostly the same diameters (monodisperse). Variance in the diameters is relatively low, such as less than ±25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.1%, 0.01%, and other values.
If each sphere has a radius ‘r’, then the length ‘a’ (see
The minimum pore size between the spheres is:
The volume of interstitial space in each unit cell is:
The ratio of interstitial volume to unit cell is a constant 47.0% for all sizes of bead particles.
The wettable surface area within each unit cell is:
2·4πr2 Eqn. (4)
A table of some of these values with respect to particle diameter is provided below.
8 × 1013
5 × 1013
1 × 1013
3 × 1012
Different packing configurations, such as body centered cubic (BCC), face centered cubic (FCC), hexagonal close packed, etc. can form in various parts of the silica colloidal crystal. Imperfections and voids can result in microcracks in some areas of the structure.
The polymer may be activated in order to immobilize certain analytes. This activation can be triggered by using electromagnetic radiation, such as ultraviolet light, infrared light, or visible light.
The shells have an uncompressed thickness 1506. Thus, the distance between the silica core of the particles when the shells are uncompressed is 2 times thickness 1506. However, the compression in packing the particles together results in a distance 1507 between the silica core of the particles. Interstitial spaces are shrunk by the amount that the uncompressed portion of the shell intrudes as well as the Young's modulus deformation of resilient material from directly between the particles laterally into the interstitial space. In other words, the shell material slightly squeezes into the interstitial space, lessening the pore size.
The compressibility of the shells can be used for fine tuning of pore sizes after the silica colloidal crystal structure is assembled. For example, the sides and top of the structure can be compressed with a mechanical or electrical actuator, causing the voids to shrink by a small amount. Likewise, stretching the structure from the sides can increase pore size.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications, websites, and databases cited herein are hereby incorporated by reference in their entireties for all purposes.
This application claims the benefit of U.S. Provisional Application Nos. 61/970,818, and 61/970,857, both filed Mar. 26, 2014, the disclosures of which are incorporated by reference in their entireties for all purposes. In addition, this application incorporates by reference the disclosure of U.S. patent application Ser. No. 14/668,485, filed Mar. 25, 2015 in its entirety.
This invention was made with government support under CA161772 and GM112387 awarded by the National Institutes of Health. The government has certain rights in the invention.
Number | Date | Country | |
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61970818 | Mar 2014 | US | |
61970857 | Mar 2014 | US |