Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase

Definitions

• the present disclosure relates generally to a multi-dimensional analytical strategy and system for analyzing antigens and particularly to an affinity selector based recognition and quantification system and method for analyzing multiple analytes in a single analysis.
• a fundamental component of both RIA and ELISA is the use of immobilized antibodies to achieve antigen selection from samples in the first step of an assay. Since the inception of these approaches, analytical immunologists have understood that binding antibodies at a surface introduces significant kinetic limitations. For instance, antigens have to travel substantial distances in terms of molecular dimensions to reach surfaces, thereby adding to the amount of time needed for antigen binding to occur in a test tube or microtiter well. Moreover,
• DMS US 51485694vl all antibodies used in an assay are bunched together at the surface of an assay well or on a particle, while antigens are uniformly distributed throughout the solution.
• incubation times of a day or more are typically used to allow time for the antigens to diffuse to the walls of the well where the antibodies are bound.
• Efforts to minimize the diffusion problems in the case of RIA involved using large numbers of very small inorganic particles to which antibodies were immobilized. Over the course of the past several years, many types of mixing, flow, heating, and even sonication procedures have been used to minimize the diffusion problem noted above. Despite these efforts, diffusion problems still exist.
• the solution becomes crowded with so many particles that the antigens must diffuse around particles not carrying their antibody.
• One proposed solution for dealing with these limitations is to immobilize multiple antibodies on a single particle.
• this solution does not completely address the issues of diffusion and stoichiometric control.
• the dilution of antibody concentration on the particle surface means that the antigens can strike particle surfaces, while not contacting their antibody.
• the concentration of an antibody needed to carry out an analytical measurement is 10 ug/mL and when 100 antibodies are to be added to a plasma sample, the total increase in protein concentration would be about 1 mg/mL. Similarly, if the concentration of protein in the plasma sample were 75 mg/mL, the increase in protein concentration would be about 1.3%. As such, it can be concluded that the addition of 100 antibodies to plasma to carry out a 100-fold multiplexed analysis would have almost no effect on protein concentration, solution viscosity, and ultimately analyte diffusion. Moreover, adding a thousand antibodies would only add 10 mg/mL of mass, or a 14% change in protein concentration; which again, would likely not be enough to impact the analysis.
• the present disclosure overcomes or ameliorates at least one of the prior art disadvantages discussed above or provides a useful alternative thereto by providing a novel multi-dimensional analytical strategy and system for analyzing antigens.
• a multi-dimensional analytical strategy for antigen analysis is provided.
• antigens in a sample solution are sequestered by antibodies in a soluble immune complex during the first dimension of analysis.
• the antibodies being added to the solution at the beginning of the analytical process are for the specific purpose of binding antigens for qualitative and quantitative analyses in subsequent steps of the multi-dimensional process.
• Cross-reacting antigens, non-specifically bound substances, and species that associate secondarily with antigens may also adsorb to immune complexes in the first dimension and are eliminated in later dimensions of analysis.
• Fractionation based on either of these two features is orthogonal to epitope :paratope recognition, and discrimination in the third and fourth dimensions is achieved in a combination of ways ranging from analyte specific chemical modifications (such as derivatization or proteolysis) and size discrimination along with adsorption and differential elution from surfaces (such as those on reversed phase or ion exchange media) or a combination of sizing exclusion and hydrophobic adsorption (as with restricted access media).
• the particular discrimination mechanism chosen, and the order in which they are coupled, depends on the chemical nature of the analytes being targeted and the fractionation mechanisms used in the first two dimensions.
• the analytes can be resolved according to their mass in the fifth dimension, collision induced dissociation of analytes in a sixth dimension, and mass analysis of the resulting fragment ions in a seventh dimension.
• a multi-dimensional method for simultaneously analyzing multiple analytes within a sample solution comprises adding affinity selectors to the sample solution to form immune complexes between the affinity selectors and the analytes;
• a multidimensional method for simultaneously analyzing multiple analytes within a sample solution comprises adding affinity selectors to a sample solution containing analytes to be measured, the affinity selectors having an affinity for one or more of the analytes within the sample solution; allowing immune complexes to form between the affinity selectors and the analytes; partially or totally resolving the formed immune complexes from non-analyte substances within the sample solution; dissociating the resolved immune complexes; separating the analytes and the affinity selectors of the dissociated immune complexes from one another by capturing the analytes through a surface adsorption process; transferring the captured analytes to a detection means; and resolving the analytes with the detection means in accordance with their mass-to-charge ratios.
• the formed immune complexes are totally or partially resolved by separating the analytes or formed immune complexes according to their hydrodynamic volume, targeting a unique structural feature of the affinity selectors or the analytes to capture antibodies of the analytes or formed immune complexes, hybridizing oligonucleotides or adsorbing and capturing biotinylated affinity selectors with immobilized avidin.
• the analytes and affinity selectors within the sample solutions are separated from one another by separating the analytes or formed immune complexes according to their hydrodynamic volume, adsorbing and differentially eluting the analytes from a hydrophobic surface, targeting a unique structural feature of the affinity selectors or the analytes to capture antibodies of the analytes or formed immune complexes, hybridizing oligonucleotides, capturing biotinylated affinity selectors with immobilized avidin, adsorbing and differentially eluting the analytes from a charged surface, adsorbing and differentially eluting the analytes from an immobilized metal affinity chelator, or adsorbing and differentially eluting the analytes from a boronic acid rich surface.
• the analytes under analysis in accordance with the present teachings may include, but are not limited to, at least one of analyte fragments, analyte derivatives and analyte isotopomers.
• the affinity selectors of the present teachings may include, but are not limited to, at least one of an antibody, an antibody fragment, an aptamer, a lectin, a phage display protein receptor, a bacterial protein and an oligonucleotide.
• the bacterial proteins can include at least one of G proteins, A proteins and proteins that are produced by an organism targeting a protein from another organism, while the oligonucleotide can include at least one of R A, DNA and PNA.
• the analytes and the affinity selectors are separated from one another by targeting a unique structural feature of the affinity selectors with an immobilized antibody.
• the unique structural feature being targeted may include, but is not limited to, at least one of a distinctive natural structural feature of the affinity selectors, a hapten that has been conjugated to the affinity selectors and an immunogen conjugated to the affinity selectors.
• the analytes are resolved by using a detection means that is configured to perform the integrated steps of: (a) ionizing analytes that have been separated according to their mass-to-charge ratio; (b) generating fragment ions of parent ions from step (a) by utilizing a gas phase fragmentation process; (c) separating the generated fragment ions from step (b) according to their mass-to-charge ratio; and (d) recording a produced relative ion current as the generated fragment ions from step (c) collide with a detector surface.
• the detection means that is used to resolve the analytes in accordance with certain embodiments, the analytes are detected by one or more of the following techniques: mass spectrometry, absorbance, fluorescence and electrochemical analysis.
• isotopically coded internal standards can be used to achieve relative or absolute quantification of the analytes under analysis.
• competitive binding assays can also be used to achieve the relative or absolute quantification of the analytes.
• antibody concentration can also be considered to collect an aliquot of an analyte from the sample solution.
• the collected aliquot is configured to fit an optimum detection range of a device that is being used to resolve the analytes.
• a method for analyzing multiple analytes within a sample solution using a plurality of orthogonal separation dimensions is provided.
• the method comprises adding affinity selectors to the sample solution to form immune complexes between the affinity selectors and the analytes and to independently sequester one or more antigens and interfering substances within the sample solution; using a selective adsorption technique to remove in a first or second orthogonal separation dimension, irrespective of order, the sequestered one or more antigens and interfering substances; providing a first separation means to partially or totally resolve the formed immune complexes from other non-analyte substances within the sample solution; providing a second separation means to partially or totally resolve the analytes from one another within the sample solution; and resolving the analytes according to their mass-to- charge ratio.
• a method for simultaneously analyzing analytes within a sample solution comprising adding affinity selectors to the sample solution to form immune complexes between the affinity selectors and the analytes; separating the formed complexes from other non-complexed substances within the sample solution by providing a first chromatographic column; dissociating the formed complexes; separating the analytes from the affinity selectors by capturing the analytes through surface adsorption of a second chromatographic column; transferring the captured analytes to a third chromatographic column that is coupled to the second
• the non-complexed substances comprise substances that do not have an epitope targeted by the added affinity selectors.
• the chromatographic column used to separate the formed complexes from other non-complexed substances within the sample solution is selected from at least one of a size exclusion chromatography column, a restricted access media column packed with a sorbent, an antibody column that is configured to target a general class of the added affinity selectors, a protein A or G column that is configured to target all of the added affinity selectors, a DNA oligonucleotide column having immunobilized DNA that is complementary to an oligonucleotide attached to one or more of the added affinity selectors, an avidin column that is configured to target biotin attached to one or more of the added affinity selectors and a chromatography column that is configured to select a naturally occurring or synthetically created feature of the added affinity selectors.
• chromatographic column used to separate the analytes from the affinity selectors by capturing the analytes through surface adsorption is selected from at least one of a reversed phase chromatography column, a restricted access media column, an immunosorbent, an immobilized metal-ion affinity chromatography column, an ion exchange column and a chromatographic retention mechanism.
• the affinity selectors include, but are not limited to, aptamers, protein A, protein G, phage display proteins, natural receptors, lectins, DNA, RNA, synthetic affinity reagents, or some other species that shows an affinity for the analyte to form a plurality of intermolecular complexes between the affinity capture agents and the analyte.
• a multidimensional method for simultaneously analyzing multiple analytes within a sample solution includes: adding affinity selectors to a sample solution containing analytes to be measured, the affinity selectors having an affinity for one or more of the analytes within the sample solution; allowing immune complexes to form between the affinity selectors and the analytes; partially or totally resolving the formed immune complexes from non-analyte substances within the sample solution in a first dimension of separation using a selective adsorption technique; dissociating the resolved immune complexes; separating the analytes and the affinity selectors of the dissociated immune complexes from one another in a second dimension of separation using a selective adsorption technique; and resolving the analytes in accordance with their mass-to-charge ratios.
• FIG. 1 is a multi-dimensional scheme showing the simultaneous analysis of multiple analytes based on an affinity selector complexing with an analyte to form a complex in accordance with the teachings of the present disclosure
• FIG. 2 is a restricted access media (RAM) particle in accordance with the teachings of the present disclosure
• FIG. 3 is an illustration of a semipermeable surface (SPS) support in accordance with the teachings of the present disclosure
• FIG. 4 is an analytical protocol for direct mass spectrometry (MS) analysis of proteins captured from a complex sample matrix by an affinity selector in accordance with the teachings of the present disclosure
• FIG. 5 is an illustration of a restricted access column in which the interior is a hydrophilic gel and the exterior is coated with immobilized trypsin in accordance with the teachings of the present disclosure
• FIG. 6 depicts the synthesis of the coating involved in the trypsin-RAM column in accordance with the teachings of the present disclosure
• FIG. 7 is an analytical protocol for a mass spectrometry analysis of proteins captured from a complex sample matrix by an affinity selector and then subjected to proteolytic digestion before further analysis in accordance with the teachings of the present disclosure
• FIG. 8 is an exemplary valving system that allows high pressure to be applied to an immobilized enzyme column in the stopped flow mode of analysis in accordance with the teachings of the present disclosure
• FIG. 9 is an illustration of a protocol used in the analysis of haptens and peptides captured by a polyclonal antibody affinity selector in accordance with the teachings of the present disclosure
• FIG. 10 is an illustration of a protocol used in the analysis of haptens and peptides captured by a biotinylated affinity selector in accordance with the teachings of the present disclosure
• FIG. 11 is an illustration of a protocol used in the analysis of haptens and peptides captured by a DNA, RNA, or PNA affinity selector in accordance with the teachings of the present disclosure
• FIG. 12 is an illustration of a protocol used for fractionating immune complexes based on molecular size in accordance with the teachings of the present disclosure.
• FIG. 13 is a liquid chromatography component of an instrument platform that is capable of carrying out a high resolution analysis of an affinity selector based process for simultaneously analyzing multiple analytes in accordance with the teachings of the present disclosure.
• the present disclosure is generally related to a multidimensional analytical strategy and system for analyzing antigens.
• one discriminating feature of the present teachings is the ability to form large numbers of inter-molecular complexes with analytes in sample solutions in a first dimension, followed by some type of inter-molecular complex separation in a second dimension that will generate fractions for further fractionation or chemical reaction in a third dimension followed by very specific types of separation or chemical reactions in a third dimension.
• FIG. 1 a scheme for simultaneous analysis of multiple analytes based on an affinity selector (S*) (such as an antibody, aptamer, lectin, protein G, protein A, phage display protein, or binding protein) complexing with an analyte (A) in the first dimension of analysis to form a complex (S*:A) in accordance with the present disclosure is shown.
• S* affinity selector
• affinity selectors While a specific affinity selector will generally be used for each analyte in accordance with certain aspects of the present disclosure, in other aspects, there can also be affinity selectors that form a complex with multiple analytes, such as would be seen with an antibody targeting the Lewis x antigen that is coupled to many glycoproteins.
• the affinity selection dimension processes in this dimension are based on complexation of the affinity selector (S*) with an analyte.
• S* affinity selector
• the symbol (*) on S indicates a unique structural feature of the affinity selector, or complex that may be exploited in selecting it later in a higher dimension.
• S* is an affinity selector such as an IgG or IgM antibody, a lectin, a binding protein, a DNA specie, an RNA specie, or any type of specie with a binding affinity for an analyte (A);
• S*:A is the non-covalent complex formed by the association of the affinity selector (S*) and a specific analyte (A).
• a specific affinity selector S* can form a complex with a single analyte or with multiple analytes depending on the selectivity of the affinity selector.
• An analyte can be an antigen, hapten, or any analyte being measured that has an affinity for S*.
• An S*:A complex will be formed for each analyte. Placing S* or A in brackets indicates their concentration in moles per liter.
• KM is the binding constant of a specific affinity selector for a specific analyte (Ai).
• the binding constant is equal to the rate of complex formation divided by the rate of dissociation.
• Each type of complex formed in solution will have such a binding constant and
• a n is any specific analyte.
• a large binding constant e.g., greater than about 10 6 .
• having the complex stay intact during separation from other, non-antigen components in the sample is an important factor to be considered. It should also be noted that while complexes with low binding affinities can be analyzed in the second dimension, it is helpful if the separation is achieved quickly and before the complex has time to dissociate.
• an affinity selector such as an antibody and an analyte (A)
• affinity reagents that do not promote high levels of cross-linking, or perhaps any cross-linking at all, can be used in accordance with these embodiments.
• a sample undergoing analysis is fractionated before immune complex formation or before affinity capture of the immune complex on a solid phase sorbent.
• the function of the optional step is to remove cross-reacting species, differentiated between natural complexes of which the antigen is a component, differentiate between isoforms of the antigen, or recognize fragments of an antigen. Removal or cross-reacting species before affinity capture of the S*:A complex (shown in Formula 1) greatly facilitates analyte analysis and can be achieved in two ways.
• cross-reacting species when they have some distinguishing feature (e.g., a unique epitope, charge property, or hydrophobicity), they can be selected from the sample either before S*:A complex formation or after S*:A complex formation, but before capture of the complex on an affinity sorbent.
• some distinguishing feature e.g., a unique epitope, charge property, or hydrophobicity
• the antigen is in several different complexes such as
• S*:A:Pi, S*:A:P 2 , S*:A:P3, and S*:A:P4, and P is one more non-analyte protein, as is common in the interactome or when an antigen is partially or totally complexed with an auto-antibody. This is sometime the case with thyroglobulin in plasma. Moreover, one of these forms may interfere with analysis of the form of antigen associated with a disease. Differentiation between these complexes is achieved by targeting non-analytes in the complex with an immunosorbent that removes one or more forms of the complex before S*:A complex formation or before the S*:A complex is captured. Removal of interfering complexes is achieved with either an immunosorbent that removes one or more forms of the complex before S*:A complex formation or before the S*:A complex is captured. Removal of interfering complexes is achieved with either an immunosorbent that removes one or more forms of the complex before S*:A complex formation or before the S*:A complex is captured. Removal
• adsorbent such as an ion exchanger, an immobilized metal affinity chromatography sorbent, a hydrophobic interaction column, or a size exclusion chromatography column than differentiates between the interfering non-analyte and analyte.
• this step is to differentiate between the native form of antigen and fragments that are of lower molecular weight. It can be the case that either a fragment or the native form of the antigen is the desired analyte being targeted for detection.
• differentiation can be achieved either with an immunosorbent or a size exclusion column, and antigens can exist in post-translationally modified isoforms.
• the matrix is of high specific surface area (area/unit volume) and the distances at any point in the solution to the surface are 10 um or less.
• the surface of the matrix should also be rich in functional groups that react readily with derivatizing agents to allow covalent attachment of reagents such as some type of binding agent that targets the tag (*) on the affinity selector. The nature of these binding agents is described below. Examples of matrices are silica and organic resin particles used in
• Chromatography particles with desirable properties will be ⁇ 20 um in size, have pores of about 100 to about 500 nm in size, and a surface area exceeding about 40 m 2 /mL.
• the material in a monolith will be silica or organic resin with through pores of ⁇ 10 um and a second set of pores between about 100 and about 500 nm.
• the symbol (*) on S* indicates a unique structural feature of either the affinity selector or complex that may be exploited in binding it to a sorbent matrix.
• the feature may be a structure element in S* alone or a new feature created as a result of S*:A complex formation.
• This unique structural feature can occur in S naturally or it can be chemically conjugated to S as a tag.
• An example of a natural feature would be amino acid sequences in a mouse, rat, rabbit, bovine, porcine, or equine antibodies that are uniquely different than those found in human immunoglobulins.
• Using an anti-mouse IgG immunosorbent attached to the matrix M would make it possible to select mouse antibodies from human plasma along with the antigens to which they have formed complexes.
• semi natural tags can be added through genetic engineering that would allow the generation of amino acid sequence tags during expression of a polypeptide affinity selector.
• One such example in accordance with this aspect of the disclosure is a single chain antibody with an added peptide tail.
• affinity selectors can be extracted from samples either alone or with analytes to which they have complexed by covalently attaching a biotin, a hapten, a highly charged group, or an oligonucleotide tag to a selector (S*).
• S* selector
• the affinity selectors can be separated from the analytes by one or more of the following non- limiting techniques: separating the analytes or formed immune complexes according to their hydrodynamic volume, adsorbing and differentially eluting the analytes from a hydrophobic surface, targeting a unique structural feature of the affinity selectors or the analytes to capture antibodies of the analytes or formed immune complexes, hybridizing oligonucleotides, capturing biotinylated affinity selectors with immobilized avidin, adsorbing and differentially eluting the analytes from a charged surface, adsorbing and differentially eluting the analytes from an immobilized metal affinity chelator, and adsorbing and differentially eluting the analytes from a boronic acid rich surface.
• anti-antibody immunosorbent media is used to capture mouse or rabbit antibodies that have been added to human plasma samples. During the course of recapturing these antibodies from the samples, any substance with which they had formed a complex will also be captured as well. After capturing the S*A complex, extensive washing of the surface bound complex is used to remove all other weakly bound components from the sample.
• This technique can be referred to more generally as the anti-affinity selector approach.
• an antibody targeting any type of affinity selector can be used to pull complexes out of samples.
• antibody targeting protein can be used to isolate S*:A complexes in which S* is an immunoglobulin. This method is very similar to the anti-antibody strategy discussed above.
• avidin sorbents are used to select biotin tagged affinity selectors and their complexes back out of samples. Again, extensive washing is used to remove substances from the sample that bind with low affinity.
• affinity selectors could also be tagged with a hapten for which an antibody has been prepared.
• a second dimension capture matrix is used in the isolation of S* :A complexes.
• affinity selector is an aptamer it can be selected from samples through the use of an
• M second dimension matrix
• the sequence on the aptamer targeted by the immobilized sequence is not used in complex formation and must be free to hybridize with the complementary sequence on the sorbent surface.
• Affinity selectors tagged with DNA, RNA, or peptide nucleic acid (PNA) oligomers will be capable of binding to a complementary DNA, RNA, or PNA sequence on the matrix M through base pair hybridization.
• Each affinity selector (such as an antibody) is tagged (coded) individually or in groups with a unique DNA sequence of 8-12 bases.
• each coded affinity selector that contacts M to which a complementary oligonucleotide sequence has been attached will bind by hybridization, while complementary DNA sequences immobilized on the sorbent can either be distributed homogeneously or spatially grouped, depending on the manner in which they will be eluted.
• Complementary oligonucleotides are placed at different locations in a column when eluted sequentially by either denaturing the complex, dissociating the complementary hybrid, or both. When they are grouped together, a more elaborate sequential release procedure must be used involving thermocycling.
• the matrix shows differential permeability to the S*:A complex instead of adsorbing it.
• a size discriminating separator such as a size exclusion chromatography (SEC) matrix, restricted access media (RAM), or semipermeable surface (SPS) media, field flow fractionation (FFF), hydrodynamic chromatography (HDC), or a membrane filtration system.
• the macromolecular S*:A complexes will have a much higher molecular weight than the low molecular weight components in samples and will be easily differentiated by size separating systems, including restricted access media (RAM) and semipermeable surface (SPS) columns. Low molecular weight, hydrophobic substances entering the pores or the semipermeable surface of these media would be differentially adsorbed. This means the RAM and SPS columns are separating molecules by both a size and hydrophobic interaction mechanism.
• RAM restricted access media
• SPS semipermeable surface
• RAM supports are generally an aggregate of submicron silica particles in which the interior surfaces of the support are covalently derivatized with stearic acid and the exterior surfaces of the support are derivatized with a glycerol ether of the structure ⁇ OCH 2 ⁇ CH(OH) ⁇ CH 2 0H.
• the average pore diameter between the submicron particles is about 6 nm on the average, which precludes entry of most proteins exceeding between about 20 to about 40 kD in molecular weight.
• peptides and haptens of less than about 3 kD readily enter the interior of a RAM support where they are adsorbed from water.
• Electrophoretic separations of complexes can be achieved by either differences in electrophoretic mobility or isoelectric point.
• FFF Field flow fractionation
• FIG. 3 an illustration of a semipermeable surface (SPS) support is provided.
• SPS semipermeable surface
• the final component of the second dimension separation process requires that either the S* :A complex or the analyte alone be released from a sorbent in the second dimension and/or transported into the third dimension for further analysis.
• the analyte can either be transported directly into the third dimension or the complex dissociated before the analyte is transported into the third dimension.
• Dissociation in the case of molecular sizing separations can be achieved by adjusting the pH at the exit from the molecular sizing column with a relatively acidic (pH 2.5) or basic (pH 12) mobile phase.
• dissociation of immune complexes from immunosorbents is achieved either by relatively acidic (pH 2.5) or basic (pH 12) conditions.
• Ab Ag complexes are dissociated as well.
• Biotinylated affinity selector:analyte complexes can be released from a mono- avidin affinity column in several ways. One is by adding biotin to the solution above the capture agent or to the mobile phase in the case of chromatography columns. This will release the biotinylated S*A complex. Mono-avidin columns can be used for many cycles when eluted in this manner. An alternative elution strategy is to apply an acidic solution having a pH of about 2.5. This will reversibly denature mono-avidin and release both the affinity selector and probably the analyte from the affinity selector.
• the affinity selector can be a polynucleotide or have a covalently attached oligonucleotide.
• complementary oligonucleotide sequences on some type of sorbent matrix (M DN A or M RN A in the Formula above) are used to adsorb the S*:A complex from the sample. Dissociation and/or release of the complex or the components of the complex from M DN A is achieved with eluents that i) are of low ionic strength, ii) are very basic, or iii) contain a denaturant.
• Dissociation and release can also be obtained by elevating the temperature to melt the DNA:DNA, R A:RNA, DNA:RNA, PNA:DNA, or PNA:PNA hybrid. Still another way would be to add a solution in the dissociation step that contains the same DNA (or RNA) sequence as in the tag on the affinity selector and then stopping the flow of the reagent across the surface of M. After flow is arrested, temperature of the solution is elevated to the point that the DNA:DNA, RNA:RNA, or DNA:RNA hybrid is melted. Thereafter, the temperature is allowed to return to room temperature so that rehybridization can occur. When the concentration of the complementary oligonucleotide added is high, it will out-compete the oligonucleotide tag on the S*:A complex, thereby leaving it in solution. The released complex may then be transported into the second dimension for further analysis.
• Analysis in the third dimension in accordance with the teachings of the present disclosure depends on the ultimate detection method to be used at the end of the analytical process, as well as whether the analytes are i) small molecules such as haptens or peptides ii) proteins, glycoproteins, lipoproteins, polysaccharides, polynucleotides, or iii) some other macromolecular species.
• FIG. 4 is a multi-dimensional "analysis option tree" (AOT) for the analysis of proteins beginning with affinity selector binding of analytes in solution. Proceeding on from affinity complex formation, in accordance with certain embodiments of the present disclosure, the number of options for analysis can increase to eight, or even more by the fifth dimension of analysis. AOTs generally begin with affinity selection in the first dimension, proceed on through a series of chromatographic and/or chemical modification steps in the intermediate dimensions of analysis, and conclude with detection by mass spectrometry, fluorescence, or electrochemical means. The type of analyte being detected, sensitivity requirements, and available
• FIG. 4 An AOT for direct MS analysis of proteins sequestered from a complex sample matrix by some type of affinity selector, generally an antibody, Fab fragment of an antibody, single chain antibody, or phage display protein is seen in FIG. 4.
• affinity selector generally an antibody, Fab fragment of an antibody, single chain antibody, or phage display protein
• FIG. 4 After the intermolecular affinity selector:analyte complex is captured from the solution in the second dimension by a sorbent targeting the affinity selector, the anti-selector:selector:analyte complex is dissociated and the components further fractionated by either size exclusion chromatography (SEC) or reversed phase chromatography (RPC) in dimensions 3 and 4 before being sent to either an ESI-MS/MS or MALDI-MS/MS for direct analysis of the intact protein.
• SEC size exclusion chromatography
• RPC reversed phase chromatography
• the outer branches of AOT for direct analysis of protein in FIG. 4 shows that the final choice is detection by either electrospray ionization (ESI) or matrix assisted laser desorption ionization (MALDI) mass spectrometry (MS).
• ESI electrospray ionization
• MALDI matrix assisted laser desorption ionization
• MS mass spectrometry
• Another element of the AOT is whether the affinity selector (Ab) will be removed from samples in dimension 3 before they are sent to the MS or left in the sample. Still referring to FIG. 4, while antibodies are removed in analytical routes A, B, C, and D, they are left in the samples of routes E, F, G, and H. As should be understood and appreciated herein, whether antibodies are removed from samples before the analysis depends on several issues. For instance, as antibodies are typically at least about 160 kD in size, when the molecular weight of the analytes is ⁇ 100 kD a MALDI-MS analysis would have no problem differentiating between the two.
• routes E and F are a high resolution, gradient eluted analytical column that is needed in routes E and F or whether a short, low resolution column would suffice as in routes G and H.
• routes E and F would be chosen.
• routes G and H are likely used.
• immobilized trypsin can also be used in a chromatography column, but in a different manner than previously described.
• the pH optimum of trypsin is in a pH range of from about 7 to about 9.
• This size excluding column is filled with trypsin digestion buffer.
• proteins readily move ahead of acidic elution buffer into the trypsin digestion buffer where proteolysis begins to occur. With proteolysis, peptides are formed that can enter the pores of the restricted access column, but they and their protein parents have moved beyond the acidic elution buffer.
• FIG. 5 shows an illustration of a restricted access column in which the interior is a hydrophilic gel and the exterior is coated with immobilized trypsin.
• the pore diameter of these particles is in the range of 6 nm or less.
• This column serves the function of achieving buffer exchange and proteolysis in the same operation.
• protein in the sample is precluded from entering the pores and migrates ahead of the buffer entering the pores of the particles.
• Proteins migrate into a region in the column filled with a buffer suitable for proteolysis. At this point, proteolysis is catalyzed by trypsin covalently coupled to the particles.
• the column When trypsin is immobilized on a porous particle medium ranging from 6 to 30 nm, the column also functions as a size exclusion column.
• the pores are of a size that will partially exclude many proteins from penetrating the immobilized enzyme matrix. Starting with 6 nm pore diameter silica particles, these particles are coated with gamma-glycidoxpropyl trimethoxysilane and the attached oxirane hydrolyzed to yield a diol. Acrolein will be
• the exclusion of proteins (and to a large extent, peptides) from the pores of the particle are both unique features of the present inventive matrices.
• Residence time of proteins in the immobilized enzyme column is controlled by either the flow rate through the column or by interrupting flow through the column at some point after proteins have entered the immobilized enzyme column and before they exit.
• This column will generally be used in the 3rd dimension of analysis as seen in FIG. 7.
• FIG. 7 shows the capture and isolation of intact proteins from mixtures after which they are converted to peptides for identification and quantification by mass spectrometry.
• Immune complexes are again isolated from samples and enriched on an immunosorbent that targets epitopes unique to the antigen targeting antibody or on an affinity sorbent targeting a tag on the capture antibody. After washing to remove substances bound to complexes with low affinity, non-covalent complexes are dissociated with an acidic ( ⁇ pH 2.5) mobile phase and antigens along with the capture antibody are transported to the immobilized trypsin column.
• proteins and acidic buffer pass through this column they are resolved as proteins migrate into a trypsin digestion buffer. During this process, proteins are converted to peptide cleavage fragments in the 3rd dimension of analysis. These fragments are reconcentrated in the 4th dimension, further resolved by some type of hydrophobic interaction chromatography, and then finally identified and quantified by either ESI-MS/MS or MALDI-MS/MS. Proteins generally yield thirty to several hundred peptide fragments, any one of which can be used to identify and quantify a protein parent. Peptides chosen for identification and quantification are generally those that have high ionization efficiency, provide a sequence that is unique to the parent protein, and are well retained by reversed phase chromatography columns.
• ESI-MS/MS and MALDI-MS/MS are more comparable in capability in this case. With either approach, it is possible to identify hundreds of peptides in a single analysis. Because suppression of ionization occurs by different mechanisms in MALDI-MS and ESI-MS, some peptides are detected better with some types of MS more than others.
• the rate of proteolysis in the immobilized enzyme column can be accelerated by raising the temperature, by sonication, or by increasing the pressure inside the column to -10,000 psi.
• An exemplary set-up for increasing the pressure in this respect is illustrated in FIG. 8, which specifically shows a valving system that allows high pressure to be applied to an immobilized enzyme column in the stopped flow mode of analysis.
• high pressure is thought to facilitate proteolysis by the partial denaturation of proteins.
• Proteolysis of a sample begins by switching the valve into a position such that the immunosorbent, trypsin column, and RPC concentrator are connected in series.
• the immobilized enzyme column in FIG. 8 is in the 3rd dimension of analysis in
• FIG. 7 Antigens and antibodies released from the affinity column in the 2nd dimension of FIG. 7 are transported directly into the immobilized trypsin column in FIG. 8 where the acidic eluting buffer and proteins are separated as the protein migrates into the trypsin digestion buffer. Based on the volumes of the columns in the 2nd and 3rd dimensions, the amount of solvent that must be pumped through the system to cause this separation is calculated.
• the valve is switched to the position indicted in FIG. 7. In this valve position, very high pressure from the pneumatic pump can be applied to the trypsin column at zero flow rates. Flow from the immunosorbent column directly to the RPC
• haptens and peptides are reconcentrated after their generation in the analytical process and/or after their separation from non-analytes in the second dimension.
• This process when it is carried out in a chromatography column, is often referred to as refocusing since analytes are adsorbed in a tight zone at the column inlet.
• Reconcentration and further separation of analytes can be achieved in multiple ways ranging from some form of affinity selection mechanism to ion exchange or hydrophilic interactions mechanisms, but a hydrophobic interaction mechanism is particularly useful because it is the most universal adsorption method, and at this stage, most analytes will be in water.
• affinity selector Reconcentration of analytes released from affinity selectors were achieved in one of two types of affinity selector.
• the advantage of this column is that it separates the affinity selector (generally an antibody) from haptens and peptides. Peptides and haptens penetrate into the pores of the RAM column or through the outer coating of the SPS column where they are adsorbed hydrophobically. Because the antibody is large, it cannot penetrate the pore or coating and is carried away to waste as indicated in FIGS. 8- 10.
• An alternative concentrator is a short reversed phase chromatography column of ⁇ 1 cm in length. Haptens, peptides, and protein are all captured on the column, in addition to selector antibodies. Because samples are being refocused at the column inlet, there is no need for a sharp, small, discrete injection of samples. In addition, large sample volumes can be loaded
• FIG. 9 is an illustration of the protocol used in the analysis of haptens and peptides captured by a polyclonal antibody affinity selector in accordance with the teachings of the present disclosure.
• haptens and peptides release from the affinity selector that captures immune complexes in the 2nd dimension and are then refocused and further resolved by a RAM or RPC column prior to ESI-MS/MS or MALDI- MS/MS. Refocusing and reversed phase separation occurs in the 3rd and 4th dimensions of analysis. While it is possible to go directly from the 2nd to the 4th dimension, the RAM or RPC concentrator preserves the analytical column. It is also possible, in many cases, to separate the Ab used in the first dimension from analytes in the 3rd dimension.
• One of the decisions in the analytical option tree shown in FIG. 9 is whether to use a RAM column or an RPC concentrator. While the RPC concentrator is simpler, has greater binding capacity, and is less expensive than a RAM column, RPC columns disadvantageously capture peptides, haptens, and antibodies. In addition, all antibody species elute together from RPC columns late in the chromatogram during gradient elution. This means that the antibody peaks will be large relative to analyte peaks. This is not a problem in most cases because peptides and haptens elute long before antibodies. The large antibody peak will not mask analyte peaks when this is true.
• the RAM or RPC concentrator can be gradient eluted directly into the ESI-MS or onto the MALDI-MS analysis plate.
• the peak capacity of these short columns is often no more than 50 components.
• There is no need for a higher resolution separation column such as the "analytical RPC" column. This is shown as occurring in the fourth dimension in FIGS. 9-10, but it is the same column that was loaded with sample in the third dimension.
• complex analyte mixtures will require much longer, higher resolution RPC columns.
• These analytical columns are from 10-50 cm in length and are packed with particles ranging from about 1.5 to about 5 um in diameter with an octadecyl silane (CI 8) coating.
• Analytical columns can have peak capacities of up to 600 components when slowly gradient eluted and heated to enhance mobile phase diffusion rates. Because substances are eluted from the RPC column into a mass spectrometer, coeluting peaks can be differentiated in either a first or second dimension of mass spectrometry.
• the type of mass spectrometry used in the 5th and higher dimensions depends on the complexity of the initial sample matrix, the number of analytes being analyzed, and analyte concentration. When the sample matrix is simple and fewer than 10 analytes are being examined, single dimension mass analysis of molecular weight alone in either the MALDI or ESI mode will be adequate. CID followed by further mass analysis of fragment ions in a 6th and 7th dimension allow higher confidence levels in identification but is probably not necessary.
• the type of mass spectrometer with simple samples is determined primarily on the basis of what is available when sample concentration is in the ug/mL to mg/mL range.
• a mass spectrometer such as a triple-quadrupole (QQQ) or quadrupole-ion trap (Q-Trap) instrument in the selected ion monitoring mode.
• Instruments such as these which mass analyze a particular ion for longer periods of time and accumulate greater numbers of ion counts for a specific analyte, can have a one hundred fold or greater sensitivity than rapid scanning instruments.
• the ESI-MS/MS instruments are particularly useful for the protocol of FIG. 9 because they provide a high level of discrimination. Again, when highest sensitivity is needed QQQ or Q-Trap type instruments provide the best solution.
• FIG. 10 shows an illustration of the protocol used in the analysis of haptens and peptides captured by a biotinylated or hapten tagged affinity selectors.
• mono-avidin is used in the 2nd dimension affinity selector because less severe conditions are required to release biotinylated species.
• Haptens and peptides can be released in the 2nd dimension either by biospecific displacement or affinity selector denaturation. The most gentle is with a biotin displacer.
• the disadvantage of this approach is that desorption kinetics are slow, requiring slow flow rates during the elution step. By contrast, partial denaturation with an acidic mobile phase, as described in FIG. 9, is much faster.
• FIG. 11 is an illustration of a protocol used in the analysis of haptens and peptides captured using antibodies tagged with a DNA, R A, or PNA.
• separation of the immune complex from other substances in a sample occurs in the 2nd dimension.
• Elution of captured antibodies from the 2nd dimension occurs by raising column temperature above the melting point of the oligonucleotide hybrid. When thermocycling occurs under flow, all dissociated species are swept from the column.
• Thermocycling under arrested flow in the presence of competing oligonucleotides allows differential displacement of specific antibodies.
• Elution conditions in the 2nd dimension are sufficiently mild that the immune complex may still be intact as it passes into the 3rd dimension and is captured.
• the immune complex will dissociate in the 3rd dimension as RPC or RAM columns are eluted with an acidic mobile phase containing acetonitrile. Whether the immune complex is intact or dissociated will not impact the net outcome of the analysis.
• the rest of the workflow is as described in FIG. 9.
• analytes are selected in the 1st dimension by
• affinity selector such as an antibody
• affinity selector such as an antibody
• ONT t composed of an ordered base sequence of RNA, DNA, or PNA. All the antibodies can have the same
• oligonucleotide sequence or each antibody species can be tagged with a different oligonucleotide sequence.
• the soluble complex is captured from samples by passing an aliquot of the solution through particles or across the surface of a solid support containing an immobilized oligonucleotide selector (ONT s ) with sequences
• oligonucleotide (ONT s ) bearing surface During the course of passing an aliquot of the solution across the oligonucleotide (ONT s ) bearing surface, analyte:selector-ONT t complexes will be captured by hybridization, forming a— ONT t :ONT s :selector analyte complex.
• Oligonucleotides (ONTO immobilized on the solid surface may be comingled or each can be located at spatially different sites.
• the support matrix for immobilizing ONT s is an organic resin, such as the pressure stable styrene-divinylbenzene resins used in HPLC.
• the POROS support matrices from Applied Biosystems are ideal examples of such resins.
• these resins withstand temperatures from about 80-90° C, as well as wide extremes in pH that may be used in analyte elution as described below.
• selector complexes in the 2nd dimension they must be released and eluted into the 3rd dimension.
• the elution step can be achieved in several different ways in accordance with the teachings herein.
• One exemplary method is to dissociate the hybrid by raising the temperate of the solid phase adsorbent bearing the—
• ONT t :ONT s selector analyte complex above the melting point of the— ONT t :ONT s hybrid while the mobile phase is passing across the surface and being transported to the 3rd dimension.
• The— ONT t :ONT s hybrid will dissociate and the ONT s :selector:analyte complex will be transported to the 3rd dimension.
• the ONT s : selector analyte complex will remain intact, but in some cases it will partially or totally dissociate. Whichever the case may be will be
• a second exemplary method of elution is to use extremes in either pH or ionic strength to dissociate the hybrids.
• the— ONT t :ONT s hybrid generally dissociates.
• Use of a pH 10 buffer at low ionic strength accomplishes the same thing.
• Immune complexes can also be fractionated according to molecular size (see FIG. 12). Immune complexes are fractionated in the 2nd dimension in this workflow using size exclusion chromatography.
• the pore diameter of the SEC column is in the range of about 100 to about 150 angstrom while the particle size of the exclusion matrix should be about 3 to about 5 um. Immune complexes will elute near the exclusion volume of the column in the case of the 100 angstrom pore diameter packing material and are directly transported to the RPC or RAM concentration or directly to the analytical RPC column. At the conclusion of this transfer, lower molecular weight species eluting from the SEC column are diverted to waste.
• the RPC columns are eluted in a linear gradient with a mobile phase ranging from 0.1% trifluoroacetic acid or 1% formic acid to the same concentration of acid containing 70% acetonitrile.
• This mobile phase is sufficiently acidic to dissociate immune complexes captured at the inlet of the RPC columns.
• the rationale for which type of mass spectrometry to use is the same as in other cases.
• proteins require the addition of a proteolytic dimension beyond that required in the analysis of haptens and peptides. This is generally achieved in the third dimension.
• FIG. 13 shows a liquid chromatography component of an instrument platform that is capable of providing the automated, high resolution separation of an affinity selector needed for high throughput and simultaneous analysis of multiple analytes.
• Sample preparation is initiated in an auto-sampler housed in a refrigerated chamber that minimizes microbial growth of a sample prior to analysis.
• Auto-sampler vials holding samples are of the conical bottom type to minimize sample volume.
• the auto-sampler also holds antibody solutions at a fixed concentration necessary for the analysis.
• the auto-sampler can also be loaded with additional reagents needed for reduction, alkylation, derivatization, proteolysis, internal standards addition, and diluents, any of which can be aliquoted into sample vials in any order.
• An analysis begins in the auto-sampler when a robotic syringe removes an aliquot of antibody, antibodies, or reagent from a reagent vial and adds them to a sample. Multiple reagents can be added sequentially to a sample vial as might be needed in reduction, alkylation, and proteolysis of a sample before analysis.
• a single antibody reagent vial may contain one antibody or all the antibodies necessary for a particular multiple analyte analysis. After dispensing a reagent or antibody into a sample the syringe goes to a vial in the auto-sampler containing reagent free buffer and is cleaned by fully loading the syringe with buffer multiple times and dispensing the buffer to a waste vial.
• the auto-sampler withdraws an aliquot of solution from a sample vial and loads a fixed volume sample loop on a high pressure valve in the sample loading position. The valve is then rotated to introduce the sample loop into a mobile phase flow path connecting it to a down stream column.
• the first down stream column (in the 2nd dimension of analysis) is generally an affinity column or size exclusion column that will partially or totally resolve the immune complex(es) from other components in the sample.
• an affinity column it is desirable that 20 or more column volumes of mobile phase be pumped through the column to remove substances bound to the immune complexes or column with low affinity.
• the column used in the second dimension of analysis can either be housed inside the refrigerated chamber of outside at room temperature as illustrated in FIG. 13.
• the column is operated at room temperature. Lower temperature operation is used with affinity columns when the capture agent is of low affinity and the binding constant of the capture agent is increased by going to lower temperature. At 5° C. the binding constant can be double that at room temperature.
• FIG. 13 shows that analysis in the 3rd and 4th dimension can be carried out in an oven.
• the illustration shows direct transfer of analytes from the 2nd dimension to the high resolution, analytical RPC column in the 4th dimension in FIGS. 9, 10, and 11.
• a 3rd dimension column can be added before valve B.
• the function of a heating column in accordance with these embodiments is to diminish the limitations known in the liquid chromatography literature as mobile phase and stagnant mobile phase limitations. By reducing mobile phase viscosity and increasing the rate of analyte diffusion, resolution is increased in RPC. This increases the resolution of RPC columns and the number of analytes that can be resolved.
• the first pump (PI) in the system in FIG. 13 provides solvents for the first two dimensions of separation. Solvent switching is done on the low pressure side of the pump, meaning that gradients generated with this pump are of the step gradient type.
• the pump P5 between the two chromatography systems is used to introduce mobile phases for dissociation of immune complex as would be needed when an SEC column would be used in the 2nd dimension and a RAM column in the 3rd dimension. The eluent stream leaving an SEC column would be merged with an acidic solution provided by P5 that would dissociate the complex before it reached a RAM column on valve B.
• Valve C is used to uncouple chromatography columns from the detectors and preclude transport of undesirable reagents or analytes to detectors. For example, it was noted above that large amounts of antibody will accompany samples into the RPC column and will be eluted at the end of the mobile phase gradient after all the analytes. In accordance with this particular illustrative scenario, valve C can be switched to the position where the
• affinity selector the simultaneous determination of multiple analytes to discriminate between analytes and a very large number of non-analytes through several dimensions of orthogonal analysis is important.
• the first dimension of discrimination will generally be an affinity selector: analyte complex formation in solution.
• affinity selector will be an antibody; however, it should be understood and appreciated herein that a variety of other selectors may be used as well.
• complex formation is executed in solution because it circumvents the need to immobilize large numbers of selectors and the inherent problems associated therewith.
• the second, third, and often fourth dimensions of analysis discussed above have shown how several chromatographic methods, and often a chemical reaction, can be coupled to provide still higher levels of analyte discrimination.
• the resolving power of these dimensions is such that captured antigens can be fractionated into a few hundred to a few thousand individual components. Many times this degree of resolution is sufficient, at which point antigens can be detected by fluorescence, absorbance, electrochemical, or some other means in which all analytes produce very similar characteristics for detection.
• These types of detection can be of very high sensitivity, yet still not discriminate between analytes. As such, analytes must be partially or totally resolved when they arrive at the detector.
• mass spectrometry provides another, quite different detection method that is orthogonal in its mode of analysis.
• mass spectrometers There are many types of mass spectrometers, several of which could be used in affinity selector based analyses of multiple analytes.
• the size exclusion methods described above examined the hydrodynamic volume of a substance. Multiple analytes may elute from a RAM or analytical RPC column together. Following ionization by either the MALDI or ESI process, analyte(s) are transported into a mass spectrometer where they are mass analyzed. Time-of-flight (TOF), quadrupole (Q), ion trap (IT), and hybrid forms of these instruments are used in mass analysis of analyte ions.
• TOF Time-of-flight
• Q quadrupole
• I ion trap
• mass spectrometry fractionates molecules on the basis of their mass, often to less than one atomic mass unit difference. Analytes thus analyzed (fractionated) are transported to an electron multiplier that produces an electrical signal, generally referred to as an ion current that can be used to quantify an analyte.
• CID Collision induced dissociation
• ETD electron transfer dissociation
• Fragment ions from the second dimension of mass spectrometry can be selected and further fragmented in a third dimension of mass spectrometry to gain even more specificity, but the amount of ion involved relative to the origin is small and thus sensitivity is poor.
• Mass spectrometers of choice in terms of sensitivity are those in which ions are produced and collected and/or analyzed continuously during the elution of a chromatographic peak and then further ionized and transported into the second dimension of MS for quantification.
• QQQ and IT-TOF instruments are examples. One of the great advantages of these instruments is that they collect many more ions for analysis.
• mass spectrometry based detection is not as sensitive as enzyme linked immunosorbent assay (ELISA) methods.
• ELISA enzyme linked immunosorbent assay
• current detection limits are in the range of 100 pg/mL, even when using 100 um internal diameter RPC columns before MS analysis.
• State-of-the-art ELISA is 1000 times more sensitive.
• the enormous advantage of using mass spectrometry in the 4th, 5th, and 6th dimensions of analysis, however, is specificity.
• the MS analyses are very fast.
• tandem mass spectral analysis can generally be carried out in a second or less with most instruments.
• Still another advantage is that chromatographic retention time is integrated.
• Still other detectors that may be used in accordance with the teachings of the present disclosure include, but are not limited to, laser induced fluorescence (LIF),
• LIF and EC detection generally requires that analytes be derivatized with a reagent that facilitates detection. In the case of LIF, this would be a fluorophore that exhibits excitation and emission wavelengths amenable to detection in the detector being used.
• the fluorescence tag agent must also react readily with analytes to facilitate the derivatization reaction.
• [Agi] is the initial concentration of sample antigen
• [Agi* ] is the concentration of tagged antigen added to the sample initially in known concentration
• [Ag e ] is the final concentration of sample antigen after equilibration
• [Ag e *] is the concentration of tagged antigen after final equilibration.
• the present disclosure is generally directed to differentiating between one hundred or more antigens (Ag) and tagged synthetic molecules (Ag*) simultaneously. This is enabled by liquid chromatography-mass spectrometry, which differentiates between all these species in a single analysis. As noted above, a particularly useful mode of running assays with large numbers of antigens is in the sequential addition, competitive binding assays with antibody saturation.
• the first step is to add a known amount of antibody [Abi] (or antibodies with multiple antigens) to the sample that exceeds the amount of antigen [Ag;] in the sample.
• Such step can be represented by the following equation:
• the second step of the assay is to add the tagged internal standard [Agi*] to the sample in known amount. The sum of the two antigens must exceed the concentration of the total amount of antibody targeting them, i.e. [Agi]+[Agi*][Ab] such that:
• all the analytes should be structurally different to be detected for the multiplexed assays, including the internal standards added (e.g., isotopically coded internal standards to achieve relative or absolute quantification of the analytes) to the assay solution.
• the assays will either have a different chromatographic retention time, a different molecular weight, or will fragment in a unique way in a mass spectrometer.
• Ag* will either be a 1 1 3 J C, 1 14 N, 1 1 8 0 0, or 2 H labeled version of the antigen or carry some combination of these isotopes that give the internal standard antigen a uniquely different mass than the unlabeled, natural version of Ag.
• An alternative coding strategy will be to derivatize antigens with some moiety that does not alter their antigenicity but gives them mass spectral properties that are uniquely different than Ag and structurally different from any other substance in the solution.
• Labels in the case of derivatization will frequently be heavy isotope coded and may be a universal coding agent that in one isotopic form, is used to code all antigens (Ag) and in an isotopically different version, is used to globally code all internal standard antigens added to the solution, i.e. Ag* in equation 5.
• Ag antigens
• the teachings of the present disclosure make it possible to identify and discriminate between thousands of antigens in a single analysis, thereby allowing thousands of immunological assays to be carried out simultaneously without
• [Ab:Ag]/[Ab:Ag*] ratio is measured at the end of the assay.
• large numbers of antigens are measured, i.e. Ag a , Agb, Ag c , . . . Ag n , and the antigens are capable of greatly varying in concentration, i.e. thousands of fold. In some embodiments, the same concentration is roughly used for all the antibodies, while in other embodiments, the
• concentration of antibody may be roughly and not precisely known.
• ratios of Ag to Ag* will be determined in the manner described above, and is generally through differential isotope labeling or global internal standard labeling methods.
• adding a known concentration of internal standard antigen makes it possible to determine the ratio of antigen to internal standard at the end of the assay.
• antibody concentration is matched to the optimum sensitivity range of the detection system, it is also possible to use the antibody as a sampling tool.
• an amount of sample can be selected that matches the sensitivity of the detector without taking into consideration the antigen concentration, as well as can be used to bring antigens into a concentration range that varies no more than ten fold, regardless of their initial concentration.
• antibody concentration is being used to bring analyte and internal standard concentration into the detection range of the measurement device used for quantification.
• quantification processes in accordance with the detection methods of the present disclosure are generally known in the art and therefore will not be discussed in great detail herein.
• quantification can be achieved in multiple ways depending on the ionization method. While ion current arising from the detection of ions following mass analysis is widely used for detection in mass spectrometry, the ionization efficiency of such methods varies widely between analytes. In fact, ionization efficiency can even vary in some cases with concentration and other unknown matrix components in the sample.
• mass spectrometers for isotopomer ratio quantification which are capable of sitting on an ion for long periods of time (seconds) while eluting from the RPC column are particularly useful because they allow many more ions to accumulate than with instruments that rapid scan the mass range of ions emerging from the RPC and fail to accumulate large numbers of ions for detection.
• Non- limiting examples of such instruments include the triple quadrupole and quadrupole ion trap instruments.
• the use of flow through plates equipped with a filter enables parallel processing and therefore high throughput application of the materials described herein.
• the use of spin columns as a micro-bioreactor shaped as a tube with a filter containing packing materials is another single use format applicable to the process described herein.
• the use of flow through plates equipped with a filter enables parallel processing and therefore high throughput application of the materials described herein.
• Use of magnetic beads containing the necessary packing materials enables direct application of these materials to the sample eliminating the need for transfer. Following the completion of a step these materials are then removed through the use of a magnet.
• multidimensional strategy described herein is through stepwise addition of various materials.
• Discrimination of analytes is achieved in a combination of ways ranging from analyte specific chemical modifications (such a derivatization or proteolysis).
• affinity based discrimination followed by chemical modifications is one strategy but it should be understood and appreciated that there are various orders that can also be used in accordance with the present teachings. It is frequently the case that affinity selection of peptides is performed in such a manner that enzymatic modification precedes the first dimension of separation. This enables the use of synthetic peptides to standardize the affinity selection process. In other cases the first dimension of separation is followed by enzymatic digestion prior to the second dimension of separation. This enables the detection of protein variants or modifications that exist outside the epitiope. In other cases the second dimension of separation is followed by enzymatic digestion prior to a third dimension of separation.

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