This invention relates to methods for using multiplexed immunoassays to quantify simultaneously the concentration of multiple analytes in a liquid sample.
Practical immunodiagnostic tests for in vitro detection of analytes in fluid samples are highly specific but vary in speed, sensitivity, and the degree to which they are qualitative or quantitative. They also vary widely in assay methods used, from solution based assays to those that are formulated on solid supports, such as beads, membranes, lateral flow strips, microtiter plates, biochips, and planar or semi-planar substrates such as coated slides. Quantitative immunoassays are usually associated with automated platforms designed to run high-throughput assays in a clinical environment. These systems are typically designed to measure a single analyte for each sample-test. Other assay methods such as ELISA plate and lateral flow assay can also be automated but are limited in the ability to multiplex.
Common methods used for inexpensive, qualitative testing are the dipstick and lateral flow strip formats. These assays are also typically designed to test for a single, or at most only a few, analytes per assay. Such test devices are usually designed to produce a visual signal on the solid support that can be observed by the naked eye, providing only a qualitative, or at best a semi-quantitative, test result. The sensitivity of the lateral flow format is generally maximized by increasing the strength of the visible signal, which decreases the lower limit of detection of the assay (LLD). In some cases, however, optimizing the visual signal creates an assay that is too sensitive, because the assays detect a concentration of analyte below the desired detection level, thereby leading to false positives. The sensitivity of the assay no-longer matches the concentration range over which one wishes to detect the analyte. In such cases, lateral flow assays have been adjusted by adding small increments of adjustment antibodies to offset the signal, so that a positive test signal is only observed when the analyte is present at or above a predetermined threshold.
A new generation of immunoassays is being developed to better meet the needs of clinicians and researchers. Multiplexed immunoassays are designed to measure multiple (e.g., as high as 42 or more) analytes in a single-sample test. These assays provide the potential to reduce costs and minimize sample volumes in certain volume-sensitive applications. Sample volume requirement is reduced by measuring a number of analytes in a single sample. Examples of sample-limited applications include pediatric testing in diagnostics, animal model testing in research, and the screening of serum banks to develop disease-specific biomarkers. In each of these cases, the volume of sample available for testing is limited. Microarray technology provides a promising platform for assaying multiplexed immunoassays that can potentially provide more sensitive and quantitative methods for measuring the concentration of multiple analytes within a single assay. The term “microarray” refers to a solid, planar or semi-planar substrate on which are arranged a set of microscopic spatially distinct areas, or spots, to which are attached one or more capture molecules. The intensity of the signal resulting from the assay is quantified using an optical imager.
Two parameters that are often used to describe the capability of a microarray assay are the lowest level of detection (LLD) and the working range. The LLD is the lowest analyte concentration that can be reliably quantified in the assay, and is a measure of the sensitivity of the assay. The working range, also variously referred to as the dynamic range or the assay range, is the range of analyte concentrations over which a change in concentration can be reliably measured, usually the range over which the optical signal is a linear function of analyte concentration. By way of example, an assay for the cytokine IL-8 having an LLD of 10 pg/mL and a working range of three logs provides a reliable quantitative measure of concentrations between 10 pg/mL and 10 ng/mL, but would not be acceptable to a researcher who is interested in IL-8 over a range of 500 pg/mL to 50 ng/mL. The assay would have adequate sensitivity at the low end of the working range (10 pg/mL), but would not discriminate IL-8 concentrations above 10 ng/mL and therefore would not meet the high-end requirements.
When a single analyte assay is too sensitive, the sample can be diluted to adjust the expected analyte range to the assay range. Dilution techniques are not suitable, however, in the case of a multiplexed immunoassay, which attempts independent, simultaneous measurement of multiple analytes in a single sample. By way of example, consider a simple two-plex immunoassay (two antigenic proteins detected in a single sample) having a working range of 10 pg/mL to 10 ng/mL for both analytes. Consider the application of this assay for detecting IL-7 over the range of 20 pg/mL to 2 ng/mL, and detecting IL-8 over the range of 500 pg/mL to 50 ng/mL. The working range of the assay is well suited to detect the target range of concentration for IL-7, but the targeted IL-8 concentration exceeds the upper end of the working range. Were the sample to be diluted (e.g., by 1:5), the IL-8 would be within the working range, but the IL-7 concentration would be below the low end of the assay working range (
The invention features a method of varying the sensitivity of a multi-analyte assay for individual analytes over several orders of magnitude, by adjusting the working range of a multi-analyte immunoassay for one of a plurality of analytes to be detected, without altering the low detection limit (LLD) and/or the working range of the assay for the remaining analytes. This is accomplished by adding to the solution phase of the assay one or more scaling agents, which are molecules that block the availability of a fraction of the target analyte by occupying the binding site, e.g., an epitope, on those analytes that are specifically bound by the capture molecule. In contrast to prior-art assays in which low concentrations of antibody relative to antigen are used to offset the concentration of available antigen by a fixed amount (referred to herein as “offset assays”), the scaling agent of the invention is present in high concentrations relative to analyte concentrations. The effect is to reduce the available analyte concentration by a fractional amount corresponding to a constant, referred to herein as a scaling coefficient (α) The residual amount of analyte remaining as free analyte is the quantity of analyte that is then measured. Thus, the scaling method of the invention is further distinct from prior offset assay methods in that, in the case of offsetting, the residual amount of free-floating antibodies is treated as an error and usually ignored. In the case of scaling, the residual amount of free-floating antigen is the quantity to be measured and thus exploited.
It is therefore the goal of the invention to vary the sensitivity and working range for one or more analytes in a sample independently using a controllable parameter that can discriminate between the analytes in the assay. The assay quantitatively determines the concentration of a plurality of analytes in a liquid sample in which each of said plurality of analytes is characterized by one or more assay parameters including without limitation low detection limit (LLD) and working range. The assay is based on a method which includes contacting the sample with a specifically selective binding reagent (e.g. ligands), preferably a capture reagent bound to a solid support, each of the ligands characterized in that it reversibly binds an analyte which is or may be present in the liquid sample to form an analyte/ligand complex, and in that it is specific for the analyte as compared to any other components which are or may be present in the liquid sample. Prior to or together with such contact with a capture agent, the sample is allowed to contact an amount of a first scaling agent which has a binding specificity for a first analyte of the plurality of analytes. Binding between the first scaling agent and the first analyte inhibits binding of a sub-population of the first analyte with its corresponding capture agent. Binding by the first scaling agent to the first analyte changes one or more assay parameters for the first analyte independent of the assay parameters for other analytes in the sample; that is, the scaling agent does not change an assay parameter for at least one other of the plurality of analytes.
The analyte to be detected is a compound, composition, aggregation, or other substance that can be specifically captured from a complex mixture of compounds, compositions and aggregations. That is, the analyte, or portion thereof, will be antigenic or haptenic having at least one determinant site, or will be a member of a naturally-occurring binding pair, e.g., carbohydrate and lectin, hormone and receptor, complementary nucleic acids, and the like. Analytes of particular interest include antigens, antibodies, proteins, carbohydrates, haptens, drugs, hormones, hormone metabolites, macromolecules, toxins, bacteria, viruses, enzymes, tumor markers, nucleic acids, and the like, although other types of substances may also be detected.
The term “scaling agent” as used herein is intended to include all substances which are able to bind the analyte, either directly (i.e., without an intermediate binding substance) or indirectly (i.e., with one or more intermediate binding substances forming a linkage). Preferably, the scaling agent is a specific binding substance capable of binding directly or indirectly to the analyte with a high affinity, typically being at least about 107 M−1, usually being at least about 109 M−1, sometimes being 1010 M−1, and optimally being 1011 M−1 or 1012 M−1 or greater. The scaling agent should be free from cross-reactivity with other substances that may be present in the sample or in an assay reagent. Preferably, the association between the scaling agent and analyte, e.g., when the analyte is an antigen, is based on highly specific noncovalent interactions between the antigenic determinant, or epitope, of the antigen and the variable-region domain of an antibody molecule used as the scaling agent. The scaling agent can be a monoclonal or polyclonal antibody raised against the analyte. Alternatively, the scaling agent can be a natural receptor for a biological analyte, an aptomer, or another molecule harvested or engineered for the purpose of specifically binding to an analyte. In cases where the analyte is itself an antibody, antigens or haptens recognized by the antibody can be used as the scaling agent of the invention.
The present invention is useful in assaying for a wide variety of analytes in virtually any type of sample which can be provided in a fluid form. Especially suitable are biological specimens, such as blood, serum, plasma, urine, cerebral fluid, spinal fluid, ocular lens liquid (tears), saliva, sputum, semen, cervical mucus, scrapings, swab samples, and the like. In addition, the method is suitable for industrial, environmental and food samples, such as water, reservoirs, process streams, milk, meat, poultry, fish, conditioned media, and the like. Under certain circumstances, it may be desirable to pre-treat the sample, such as by suspending in fluid, separation, dilution, concentration, filtration, chemical treatment, or a combination thereof, in order to improve the compatibility of the sample with the remaining steps of the assay. The selection and pretreatment of biological, industrial, and environmental samples prior to immunological testing is well known in the art.
Categories of immunoassays include direct and indirect competitive assays, and non-competitive assays such as sandwich assays. A competitive assay relies on competition for binding to a specific binding agent between a known amount of labeled analyte and an unknown amount of analyte in the sample. The more analyte that is present in the sample, the less binding agent available to bind to a competing analyte or analyte-analogue. In a sandwich assay, a binding agent is immobilized on a solid support to serve as a capture agent. Analyte in the test sample is allowed to react with the binding agent on the support. After the support is washed, a second, detectable agent that binds specifically to a different epitope on the analyte is added and allowed to react with the captured analyte, thereby becoming “sandwiched” between the detectable binding agent and the immobilized binding agent. After any excess detectable binding agent is washed away, observations are made of detectable binding agent bound to the sandwich complex. The observed signal is directly proportional to the amount of antigen in the test sample.
Accordingly, in one aspect, the invention features a method of adjusting the concentration of at least one but not all of a plurality of analytes in a fluid sample to match a working range of detection of an analyte assay system. The method includes the steps of a) obtaining characterization of a working range of detection of an analyte assay system for quantitative determination of the concentration of a plurality of analytes in a fluid sample, the characterization including a high end and a low end of the working range; b) providing a fluid sample in which each of a plurality of analytes may or may not be present within an expected initial concentration range having a high end and a low end, the plurality of analytes including at least one high concentration analyte having a high end expected concentration range that exceeds the high end of the working range of the assay system; and c) adjusting the expected concentration of the high concentration analyte by a proportional scaling constant, α, so that the high end of the adjusted expected concentration range is less than or equal to the high end of the working range, without adjusting the expected concentration range of at least one other of the plurality of analytes. The ratio of the adjusted concentration of the high concentration analyte to the initial concentration of the high concentration analyte can be independent of the initial concentration of the high concentration analyte.
As discussed above, one manner of adjusting the expected concentration of the high concentration analyte is by a) providing a scaling agent (S) having binding specificity for at least one, but not all, of the plurality of analytes, whereby binding of analyte by the scaling agent prevents detection of the analyte by the assay system; and b) introducing a certain amount of the scaling agent (S) to the sample to create a reaction mixture, under conditions such that, subsequent to the reaction mixture coming substantially to equilibrium binding conditions, the reaction mixture includes a scaling agent-analyte complex portion having a scaling agent-analyte complex concentration ([AS]), a free analyte portion having a free analyte concentration ([Af]), and a free scaling agent portion having a free scaling agent concentration ([Sf]), wherein the introducing results in a total scaling agent concentration ([ST]) in the reaction mixture is equal to the sum of the free scaling agent concentration plus the scaling agent-analyte complex concentration ([Sf]+[AS]), and a total analyte concentration ([AT]) in the reaction mixture equal to the sum of the free analyte concentration plus the scaling agent-analyte complex concentration ([Af]+[AS]). The total concentration of scaling agent [ST] is preferably greater than or equal to the high end expected initial concentration of the high concentration analyte in the reaction mixture. [ST] can be at least four times greater than [Af] and the product of [Af] and Ka is less than one fourth. In some embodiments, [ST] is at least nine times greater than [Af] and the product of [Af] and Ka is less than one ninth. It is understood that “equal to”, as used herein, refers to concentrations that that approach equality, and are thus approximately equal.
In one embodiment, the scaling agent can be introduced to the sample in solution form, in which case the concentrations of analyte, scaling agent, and scaling agent, complex is a function of the volume of reaction mixture resulting from the combined fluid sample and reagents. Another option is to add the scaling agent as a dry reagent, particularly in the context of an assay conducted in a biochip.
In some embodiments, the method of the invention can further include introducing the sample to a solid support including at least one immobilized capture agent, the capture agent having binding specificity for at least one of the plurality of analytes, under conditions permitting binding between the analytes and the capture agent to form analyte-capture agent complex, and determining the analyte-capture agent complex on the solid support as an indication of the initial concentration of the analytes in the fluid sample. Binding of the scaling agent to the high concentration analyte prevents binding of the high concentration analyte to the at least one capture agent. The fluid can be introduced to the solid support prior to adjusting the expected concentration of the high concentration analyte. Another option is to adjust the expected concentration of the high concentration analyte prior to introducing the fluid sample to the solid support, or the adjusting step can coincide with introducing the fluid sample to the solid support.
Preferably, the ratio of the adjusted concentration of the high concentration analyte to the initial concentration of the high concentration analyte is independent of the initial concentration of the high concentration analyte.
Preferably, where the scaling agent binds the high concentration analyte with a binding affinity Ka, and [ST] is greater than [AT], the scaling constant, α, is proportional to, or in some cases equals, (Ka[ST]+1). Under these conditions, the ratio of the free analyte concentration to the total analyte concentration, ([Af]/[AT]), is independent of the total analyte concentration ([AT]) at the total scaling agent concentration ([ST]) and is independent of the total analyte concentration within the adjusted concentration range.
The amount of analyte-capture agent complex on the solid support can be determined by measuring a parameter representative of the amount of each analyte bound to the capture agent as an indication of the initial concentration of the analyte in the sample. This can involve measuring a detectable signal bound to the support. By way of example, one or more detection binders can be introduced to the solid support under conditions permitting the detection binder to bind the analyte-capture agent complex. The amount of detection binder bound to the analyte-capture agent complex is an indication of the initial concentration of the analytes in the fluid sample. Where the detection binder includes a detectable signal, the amount of detection binder bound to the analyte-capture agent complex is determined by measuring the detectable signal, the signal being proportional to the concentration of the analyte in the sample.
In some embodiments, the scaling agent-analyte complex can be removed from the reaction mixture prior to contacting the reaction mixture with a solid support.
In other embodiments, e.g., where the assay system is a competitive assay, the method further includes the step of, prior to introducing the sample to the solid support, introducing to the reaction mixture an analyte analogue conjugate that is capable of competing with the high concentration analyte for binding to the scaling agent and the capture agent. Preferably, the analyte analogue conjugate bears a detectable label and/or emits a detectable signal. In such assays, one determines the amount of each of the analytes bound to the at least one capture agent by determining the amount of analyte analogue conjugate bound to the solid support, as an inverse indication of the amount of the corresponding analyte bound to the solid support, and as an inverse indication of the initial concentration of the corresponding analytes in the fluid sample.
Where the analyte analogue conjugate is present in the reaction mixture at a total analyte analogue conjugate concentration [A*T], the total scaling agent concentration [ST] should be greater than or equal to a sum of the total analyte concentration and the total analyte analogue conjugate concentration ([AT]+[A*T]).
Preferably, the total scaling agent concentration ([ST]) is at least four times greater than a sum of the free analyte concentration and the free analyte analogue conjugate concentration ([Af]+[A*f]), and the product Ka ([Af]+[A*f]) is less than one fourth. In some embodiments, the total scaling agent concentration ([ST]) is at least nine times greater than the sum of the free analyte concentration and the free analyte analogue conjugate concentration ([Af]+[A*f]), and the product Ka ([Af]+[A*f]) is less than one ninth.
In another aspect, the invention features a method of measuring the affinity of a binding agent, Ka, for an analyte, the method including the steps of a) providing a fluid sample including an analyte; b) experimentally determining a value of a scaling constant α; and c) calculating Ka, where Ka is equal to (α−1)/[Sf].
The invention further includes an apparatus for performing an assay for the quantitative determination of the concentration of a plurality of analytes in a fluid sample, the assay system including a defined working range having a high end and a low end. The apparatus includes a) means for providing a fluid sample in which each of a plurality of analytes which may or may not be present within an expected initial concentration range having a high end and a low end, the plurality of analytes including at least one high concentration analyte having a high end expected concentration range that exceeds the high end of the working range of the assay system; b) means for adjusting the expected concentration of the high concentration analyte by a proportional scaling constant, α, so that the high end of the adjusted expected concentration range is less than or equal to the high end of the working range, without adjusting the expected concentration range of at least one other of the plurality of analytes; c) means for introducing the sample to a solid support including at least one immobilized capture agent, the capture agent having binding specificity for at least one of the plurality of analytes, under conditions permitting binding between the analytes and the capture agent to form analyte-capture agent complex; and d) means for determining the analyte-capture agent complex on the solid support as an indication of the initial concentration of the analytes in the fluid sample. The adjusting means can include means for introducing a certain amount of the scaling agent (S) to the sample to create a reaction mixture, under conditions such that, subsequent to the reaction mixture coming substantially to equilibrium binding conditions, the reaction mixture including a scaling agent-analyte complex portion having a scaling agent-analyte complex concentration ([AS]), a free analyte portion having a free analyte concentration ([Af]), and a free scaling agent portion having a free scaling agent concentration ([Sf]); wherein the introducing results in a total scaling agent concentration ([ST]) in the reaction mixture equal to the sum of the free scaling agent concentration plus the scaling agent-analyte complex concentration ([Sf]+[AS]), and a total analyte concentration ([AT]) in the reaction mixture equal to the sum of the free analyte concentration plus the scaling agent-analyte complex concentration ([Af]+[AS]) and wherein the scaling agent (S) having binding specificity for at least one, but not all, of the plurality of analytes.
The means for determining the analyte-capture agent complex on the solid support can include means for measuring a parameter representative of the amount of each analyte bound to the capture agent as an indication of the initial concentration of the analyte in the sample. Preferably, the means for determining the analyte-capture agent complex on the solid support includes means for measuring a detectable signal. The means for determining the analyte-capture agent complex on the solid support can also include means for introducing at least one detection binder to the solid support under conditions permitting the detection binder to bind the analyte-capture agent complex, and means for determining the amount of detection binder bound to the analyte-capture agent complex as an indication of the initial concentration of the analytes in the fluid sample, such as, e.g., means for measuring the detectable signal, the signal being proportional to the concentration of the analyte in the sample.
In some situations the apparatus can further include optional means for removing the scaling agent-analyte complex from the reaction mixture prior to the contacting the reaction mixture with a solid support.
The apparatus can further include means for introducing to the reaction mixture an analyte analogue conjugate including a detectable signal, the analogue conjugate capable of competing with the high concentration analyte for binding to the scaling agent and the capture agent. Here, the apparatus can further include means for determining the amount of analyte analogue conjugate bound to the solid support as an inverse indication of the amount of analyte bound to the solid support, and as an inverse indication of the initial concentration of the analytes in the fluid sample.
Scaling an assay involves altering the LLD and working range of an assay. It is often the case with quantitative binding assays, e.g., immunoassays that the working range of quantification is not matched to the expected range of analyte. For multiplexed assays it is advantageous to scale the sensitivity and working range for one or more analytes without affecting the sensitivity and working range of the assay for other analytes in the sample. Scaling agents are added to reduce the proportion of a particular analyte in the sample, thereby reducing the effective concentration of that analyte by a constant scaling coefficient.
Accordingly, the invention relies on binding a specified amount of the analyte present in the sample by pre-exposing the sample to a scaling agent, which is a molecule having a high binding affinity for the analyte, preferably an antibody or receptor having high specificity for the target analyte to be scaled. The scaling agent binds to a portion of the analyte in the sample, and the amount of scaling agent used is equal to or in excess of a scaling coefficient that can be determined either theoretically or experimentally. Due to the equilibrium nature of association and dissociation between scaling agent and analyte in solution, the sample can be expected to maintain a finite amount of unbound, i.e., free, analyte, which is then readily detectable in the subsequent detection phase of the assay.
Preferably, the amount of scaling agent added to the sample is selected to be equal to or greater than that required to elicit a transition between a scaling portion of a partition curve and an offset portion of the partition curve, as further defined below.
In operation, the first step of the assay of the invention includes reacting a sample suspected of containing the analyte with a certain amount of the scaling agent, the certain amount selected to reduce the amount of available free analyte in the reacted sample according to a constant scaling coefficient. The scaling agent is exposed to the sample during the course of a pre-treatment step prior to introducing the assay sample to the median, device, or solid support bearing the capture agent. The scaling agent can be introduced in a suitable buffer or other liquid carrier, which is combined with the sample to form a liquid reaction phase and allowed to reach equilibrium. Alternatively, the scaling agent can be introduced on a solid phase (such as particles, beads, a dipstick, an agarose gel, or the like) which is combined with the liquid sample and permitted to reach equilibrium solution conditions. In a third alternative, the scaling agent can be bound to the interior of a transfer pipette, a filter, or a pre-treatment chamber or well used to introduce the sample to the test device, as long as the scaling agent reaches equilibrium in the sample prior to exposure to capture agent. The reaction between the sample and the scaling agent is allowed to proceed to equilibrium prior to any subsequent assay steps, usually proceeding for at least about ten seconds, more usually requiring at least one to two minutes. The time required for the scaling phase of the assay to reach equilibrium varies according to temperature and solution conditions of the medium and the strength of affinity of the scaling agent for the analyte, and can be minimized by use of scaling agents with maximum binding affinity. Preferably, the solution conditions of the assay are determined so as to assure sufficient and consistent binding conditions between scaling agent and analyte prior to performing subsequent steps of the assay.
After the reaction between the scaling agent and sample has been allowed to reach equilibrium, the steps of the detection phase of the assay are initiated, usually without separating the analyte/scaling agent complex from the reaction phase. The reacted sample is then contacted with a capture agent in the detection phase of the assay. Preferably the capture agent is immobilized on a solid support, most preferably a capture agent fixed to a microarray formatted support. In this way, residual free analyte remaining in the reaction phase (i.e., analyte that is not bound to scaling agent) binds to the capture agent on the solid support.
The degree to which a scaling agent adjusts the working range of the assay for a given analyte depends primarily on the binding affinity of the scaling agent for the analyte (taking into account the number of epitopes on the analyte that are recognized by the scaling agent) and the concentration of the scaling agent in relation to antigen concentration. For the purposes of simplifying discussion herein, the amount of scaling agent used to adjust the working range of the detection assay is best modeled by the case of a sandwich immunoassay system in which the scaling agent is an antibody, the analyte is an antigen, and there is a one-to-one binding association between antigen (Ag) and antibody (Ab) per antibody/antigen (AbAg) complex. It is within the understanding of those skilled in the art to adjust this model to accommodate multi-epitope analytes, higher binding ratios, and alternative types of complimentary scaling agent/analyte pairs.
The dynamics of the method of the invention can be modeled according to the laws of mass action, which are predictable, quantifiable and can thus be exploited to tailor an immunoassay for a particular analyte. The relative concentrations of free antibody [Ab], free antigen [Ag], and antibody/antigen complex [AbAg] in a reaction mixture at equilibrium are defined by Equation (1).
where,
The affinity constant, Ka, is characteristic of a given antibody. Low-affinity antibodies have Ka values between 107 to 109 L/mol; high-affinity antibodies have Ka values as high as 1011 L/mol/or even 1012 L/mol.
Assuming a one-to-one correspondence of antibody to antigen in the AbAg complex, the initial antigen concentration [Agi] is equal to the equilibrium free analyte concentration [Ag] plus the equilibrium concentration of the antibody/antigen complex [AbAg] according to Equation (2). Similarly, the initial antibody concentration [Abi] is equal to the equilibrium free antibody concentration [Ab] plus the equilibrium concentration of [AbAg] complex according to Equation (3).
[Agi]=[Ag]+[AbAg] (2)
[Abi]=[Ab]+[AbAg] (3)
Equations (1), (2) and (3) can be combined to show that the concentration of free antigen at equilibrium is a function of the antibody affinity constant and the initial concentrations of antibody and antigen, according to Equation (4).
where:
[Agi]=the initial molar concentration of antigen (mol/L), an
[Abi]=the initial antibody concentration (mol/L).
Equation (4) can be used to examine the ratio of free antigen to initial antigen over a range of initial antigen concentrations and in the presence of a fixed concentration of soluble antibody (
Within the scalar region at the low range of initial antigen concentration, the concentration of free antibody can be treated as a constant, and Equation (1) can be rewritten:
It follows from Equation (5) that when the free antibody concentration is constant, the ratio of bound antigen to free antigen is also constant. This means that when the scaling antibody concentration is much higher than the antigen concentration, the net effect is to reduce the antigen concentration by a constant scale factor. This relationship is exploited by the method of the invention to adjust the equilibrium concentration of free antigen to within the working range of the assay. This is illustrated by the hypothetical reaction conditions used in
The relationship between the initial antigen concentration and the initial concentration of scaling agent can be derived from Equation (1). First, Equation (3) is rearranged to get:
[Ab]=[Abi]−[Ab+Ag] (6)
Substituting into Equation (1) and rearranging yields:
Adding one to both sides and inverting yields:
Equation (8) defines the ratio of free antigen to total antigen (1/α) in terms of the initial antibody concentration and the free antigen concentration. The left hand side of Equation (8) is the fraction of total antigen concentration that remains free at equilibrium. The right hand side of the equation indicates a dependence on antibody affinity, initial antibody concentration, and equilibrium antigen concentration.
Examining the equilibrium relationship expressed in Equation (1) further suggests that when a high concentration of soluble scaling antibody is mixed with a sample having a relatively low concentration of antigen, a large proportion of the antigen will complex and no longer be available for detection by the capture agent. Due to the nature of the equilibrium between association and dissociation of the antibody/antigen complex, however, a finite portion of the antigen remains present in solution as free antigen. The ratio of the initial antigen concentration to the concentration of free antigen at equilibrium (α) is a useful parameter for modeling the effect of soluble antibody on the concentration of free antigen over a wide range of initial antigen concentrations.
This relationship is further explored in
(1) Scalar Region
The scalar region is seen on the left side of the graph in the region of relatively low initial antigen concentration, where excess antibody reduces the available antigen concentration by a constant factor, variously referred to herein as the “scalar constant”, “scalar coefficient,” or “scalar factor.” As seen on the left side of the graph in
In the scaling region at the far left of the graph shown in
[Ag]0 (9)
In the scalar concentration range in which this simplifying assumption holds true, the portion of antigen that remains free at a given concentration of scaling agent (1/α) is calculated according to Equation (8). For the assumption in Equation (9), Equation (8) reduces to:
Equations (9) and (10) are valid over the limited range of initial antigen concentrations defined herein as the scaling range.
For relatively low concentrations of antigen, Equation (10) expresses the relationship between the fraction of antigen that is free at equilibrium, initial antibody concentration, and binding affinity. Equation (10) can be used to calculate antigen depletion for a given antibody affinity and antibody concentration.
The ratio (α) is defined as the ratio of the initial antigen concentration to the free antigen at equilibrium, and is inversely related to Equation (10):
The ratio α can be empirically measured by running the same assay with and without a high concentration of scaling agent, as demonstrated by an assay of a sample containing the analyte IL-1B (
For concentrations of soluble antibody larger than the concentration of initial antigen, the scaling constant α is only a function of antibody concentration and affinity. For any given affinity, the scalar constant is determined independent of antigen concentration by choosing an antibody concentration. Within the scaling region, the coefficient α is thus determined solely by the concentration of the scaling agent and the binding affinity of the scaling agent for the analyte. For the example shown in
(2) Offset Region
At antigen concentrations that are higher than the antibody concentrations, the right hand side of Equation (8) approaches one. A high concentration of antigen relative to antibody results in little antigen depletion on a percentage basis (see
Referring again to
As discussed above, many lateral flow immunoassays are simple tests that provide qualitative positive/negative test results, and frequently are designed to provide a qualitative indication that an analyte is present in a sample only when the analyte is present above a threshold level. The assay response exhibits a relatively abrupt transition from below the threshold concentration to above it. A sharp response allows a well-defined delineation between the presence and lack of a specific analyte. On occasion, such assays exhibit a sharp transition close to, but not precisely at, the threshold of interest. If the transition occurs at a slightly lower antigen concentration than desired, then the assay developer can add a small amount of antibodies to the sample to reduce the amount of available antigen by a fixed amount, which is referred to herein as an “offset” of the concentration of antigen in the sample. For example, the assays disclosed in U.S. Pat. No. 5,541,069 and U.S. Pat. No. 5,780,308 involve adding a low concentration of soluble antibody to a sample to effectively offset the concentration of antigen by a specific amount. There is only a small, defined amount of antibody, so the majority of the antibody forms a complex with antigen, thereby blocking the antigen from detection by the assay. The result is that a fixed amount of antigen is “soaked up”, effectively removing antigen from the assay. This has been variously referred to as “blocking”, “scavenging”, or “sequestering” the analyte. The offset model of the prior art ignores the equilibrium state of association and dissociation between the antibody and the antigen in solution.
Once again Equation (4) is used to further examine the solution conditions when offsetting antigen levels with small amounts of antibody. An offset in the antigen concentration is created by adding a small amount of antibody to the sample to “soak up” a fraction of the antigen. Referring to the assay illustrated in the graph in
The offset effect illustrated in
Within the offset range, the concentration of bound Ag at equilibrium is equal to the difference between the initial and final antigen concentration (
The use of soluble antibodies by prior art offset assays can be more easily visualized by graphing the offset between the initial antigen level and the antigen level after the antibodies are added. The development of the offset as a function of increasing antigen concentration can be calculated by subtracting the equilibrium concentration from the original concentration over the assay range.
(3) Scalar to Offset Transition
Between the scalar phase and the offset phase is a transition phase, in the mid-range of initial antigen concentrations, where the presence of soluble antibody causes the percentage of free antigen to change rapidly. There are two criteria with which to define the limits between the Scalar and Offset regions. One is suggested by Equation (10) while the other is best illustrated graphically.
The boundary between the scaling region and the offset range can be defined by Equations (8), (9) and (10). The approximation of Equation (9) can be further clarified by considering the numerator and denominator of Equation (8) separately. For the approximation to hold true in the numerator, the product of the binding affinity, Ka, and the equilibrium antigen concentration must be less than one fourth, and preferably less than one tenth. For the approximation to hold true in the denominator, the initial antibody concentration must be at least four times, preferably ten times, the equilibrium antigen concentration over the expected assay range. This is summed up in the following equations:
If the conditions of Equations (13) and (14) hold true, then the resulting assay can be said to be operating within the constant scaling range of the assay.
The graph in
A second criterion by which to delineate scaling effects from offset effects is easily illustrated by viewing the scalar and offset curves on the same graph. In
The loss of scaling fidelity and the attainment of 100% offset are asymptotically approaching the same concentration for arbitrarily high antibody affinities. The limiting point is the initial antibody concentration. This suggests that the expected initial antigen concentration range relative to initial antibody concentration can be used to differentiate the scalar region from the offset region. Specifically, for initial antigen concentrations lower than the initial antibody range, the analyte concentration is scaled. For an initial antigen concentration that exceeds the initial antibody concentration, the analyte concentration is offset.
This is logically consistent with the workings of the assay. For an arbitrarily high binding affinity, all of the antibodies will complex with any available antigen. Therefore, the attainment of 100% offset will occur exactly when the antigen concentration equals the antibody concentration. Offset assays exceed this level, and therefore are practiced at initial antigen concentration ranges that exceed the initial antibody concentrations. The present invention, on the other hand, relies on the presence of a small, constant concentration of antigen. For arbitrarily high binding affinities, the consistency of the free antigen concentration (as a percentage of the total antigen concentration) loses fidelity at a concentration no higher than the initial antibody concentration. Therefore, the present invention anticipates an antigen concentration range that is always lower than the initial antibody concentration.
In general terms, it can be predicted that assays with a total antigen concentration range at or below the scaling antibody concentration operate in the scaling range, and those that have expected antigen concentrations above the antibody concentration operate in the offset domain. In other words, the full range of the “offset” assay extends well beyond the point at which the offset takes effect.
(4) Measuring Antibody Affinity
Equation (11) can be used to measure the affinity of an antibody from comparative assay data. If the scaling factor is determined experimentally at conditions of excess antibody, then the antibody affinity constant, Ka, can be calculated from Equation (15).
As discussed in reference to
(5) Antigen Availability vs. Assay Sensitivity
An interesting relationship is found by examining Equation (15). For large scaling ratios, α is roughly proportional to the antibody affinity. In other words, antigen availability is inversely proportional to antibody affinity. When the basic assay response is proportional to antibody affinity, the two effects cancel out. This can be useful for eliminating the effects of variations in scaling antibody affinity on assay response, which would be valuable in maintaining repeatability from batch-to-batch of reagents, or with the same reagents over time.
(6) Adjustment of Scaling Factor for Multiple Epitopes
Those skilled in the art will appreciate that an antigen complexes with an antibody at a particular molecular structure called an epitope, and there may be one or more identical epitopes for a given antigen. The signal resulting from an assay will generally be proportional to the number of available epitopes. One way to reduce the signal of a particular antigen, without affecting the other antigens in the assay, is to block a fraction of available epitopes on the target antigen by introducing an appropriate concentration of scaling agents for that epitope into the sample. The concentration of scaling agent required will be a function of several variables, including the number of epitopes per antigen and the magnitude of sensitivity shift desired.
Commonly, e.g., with high affinity IgG antibodies used as scaling agents in an assay for small molecule analytes (such as hormone metabolites and drugs), the valence of the scaling agent for the analyte is two, while the valence of the analyte for the scaling agent is one, i.e., it is sufficiently small that it presents only one binding site to the scaling antibody.
(7) Microarray Technology
When the microarray is exposed to a liquid sample, molecules bound to the spot interact with molecules in the liquid phase. Microarrays that serve as reaction templates for immunoassays are useful for detecting analytes in complex mixtures of proteins. To adapt an immunoassay to a microarray environment, antibodies are used as capture and/or detection agents to quantify the concentration of antigenic proteins in solution matrices such as serum or cell lysates. In the case of sandwich immunoassays, a first antibody (“capture antibody”) is immobilized to a solid support, and used to bind a specific antigenic protein in a fluid sample (the “analyte”), and thus remove the analyte from the solution phase. The remaining sample is then washed away and a solution containing a labeling agent, e.g., a detection antibody, is used to label the captured analyte. The detection antibody employs a detectable label as a signaling means (e.g. fluorescence tags) that is either conjugated directly to the antibody or becomes associated with it through an intermediate complex such as biotin/streptavidin.
The capture agent is immobilized to the solid support by methods known to those skilled in the art. Preferably the amount of capture antibody fixed to the solid support is small relative to the amount of sealing agent employed to adjust the linear range of the assay, so that the molar amount of capture antibody need not be accounted for in the equilibrium conditions discussed above.
After the addition of a sample to the microarray, analytes present in the sample bind the specific ligands. The degree of binding is measuring using an optic imager, e.g., a fluorescent imager or a chemiluminescence light reader.
(8) Additional Reaction Formats
Various analytical devices and systems suitable for performing the method of the invention are generally known to those skilled in the art. By way of example, the method of the invention can be used in conjunction with a solution based multiplexed assay, such as that provided by Aclara Biosystems, Inc. (Mountain View, Calif.). The method of the invention is also useful in association with multiplexed assays performed with capture ligands immobilized on a variety of solid supports known to those skilled in the art, such as, without limitation, micro-particles and/or beads, microtiter plates, biochips, planar or semi-planar substrates such as coated slides, lateral flow strips, and various membranes (e.g., cellulose, cellulose nitrate, cellulose acetate, glass fiber, nylon, polyelectrolyte ion exchange membrane, acrylic copolymer/nylon, or polyethersulfone). Examples of such systems include, without limitation, assays conducted in multi-well microtiter plates such as, e.g., SearchLight™ (Pierce Biotechnology, Rockford Ill.), the Multi-Array™, Multi-Spot™, Sensor™ systems (Meso Scale Discovery, a division of Meso Scale Diagnostics LLP, Gaithersburg, Md.) and the A2™ Multiplex Immunoassay using the Biomek® FX laboratory automation workstation (Beckman Coulter, Inc., Fullerton Calif.); assays conducted on or with beads as solid supports, e.g., the XMAP® system (Luminex Corporation, Austin Tex.); immunochromatographic systems based on membranes and/or lateral flow (e.g., RAMP®, Response Biomedical Corporation, Burnaby Calif.); and biochip based systems such as, e.g., the Infinit™ Analyzer and BioFilmChip™ microarray system (Autogenomics Inc., Carlsbad, Calif.) and the RX daytona and RX imola systems of Randox Laboratories Ltd (Great Britain). Additional systems that can be used in conjunction with the method of the invention include, e.g., the Stratus CS Analyzer™ of Dade Behring Inc. (Deerfield, Ill.); and systems of Bayer Healthcare Diagnostics Division; and the Triage(r) and Triage(r) Meter Plus systems of Biosite, Inc. (San Diego, Calif.), and the Access® 2 immunoassay systems, SYNCHRON LX®i 725 combination chemistry and immunoassay workstation, and the high throughput UniCel DxI 800 Access immunoassay (Beckman Coulter, Fullerton Calif.).
Suitable signal producing systems include at least one component and may comprise two or more components, including enzymes, substrates, catalysts, enhancers, and the like. In the examples herein, a detection agent is used to label the captured analyte. Usually at least one component of the signal producing system will be, or will be attached to, an agent capable of binding the analyte/capture agent complex, and will thus be bound to the solid phase in the detection phase of the assay of the present invention. Numerous suitable signal producing systems are described in the patent and scientific literature, and will be known to those skilled in the art, e.g., detection labels, imagers, and readers based on fluorescence, radioisotopes, chemiluminescence, colloidal gold, or enzymatic activity, e.g., streptavidin-horseradish peroxidase (SA-HRP). See, e.g., U.S. Pat. Nos. 4,313,734 and 5,529,902, hereby incorporated by reference. The use of a fluorescence producing system that results in attachment of a florescent tag on the solid phase support is preferred. The detection agent employs fluorescent tag, either conjugated directly to the detection agent or attached through an intermediate complex such as biotin/streptavidin. The intensity of the signal resulting from the assay is then quantified using an appropriate imager, e.g., a laser scanning confocal microscope such as a Bio-Rad Lasersharp MRC 500 (Bio-Rad Laboratories Ltd.,). Methods for the adsorption of proteins and protein analogues to colloidal gold are described in, e.g., Leuvering, U.S. Pat. No. 4,313,734, hereby incorporated by reference. Analyte analogue conjugates bearing detectable labels are also well known to those skilled in the art; see, e.g., U.S. Pat. No. 5,851,776, hereby incorporated by reference. Various apparati and devices for, e.g., sample injection, reagent delivery, temperature control, coupling of capture agents to solid supports, reaction mechanics, fluid mechanics, separation technologies, labeling and detection, imaging, calibration, data collection and analysis, are further known to those skilled in the art according to the following references, each of which is hereby incorporated by reference: U.S. Pat. Nos. 6,852,503, 6,638,728, 6,709,743, 6,437,179, 6,040,150, 6,432,662, 5,532,379, 5,538,858, 5,527,688, 5,503,741, 5,266,686, 5,112,952, 5,142,027, and 5,219,528; and US published applications US 2005/0032121, US 2004/0219622, and US 2002/0192736; U.S. Pat. Nos. 6,919,173, 6,242,236, 5,986,811, 5,717,453, 6,319,670, 6,673,533, 6,362,011, and 5,866,434; and US published applications US 2005/0142033, US 2004/00336610, US 2003/0113713, US 2003/0124572, US 2005/0052646, and US 2002/0086335; U.S. Pat. Nos. 6,773,812, 6,916,661, 6,905,766, 6,696,265, 6,649,414, 6,696,304, 6,658,357, 6,592,822, 6,046,807, 6,632,526, and 6,524,793; and US published applications US 2005/0152593, US 2005/0151964, US 2005/0073686, US 2005/0030519, US 2004/0039201, US 2001/0026920, and US 2005/0118574; U.S. Pat. Nos. 6,818,399, 6,673,550, 6,649,351, 6,627,400, 6,306,273, 6,284,113, 6,103,537, 6,103,199, 6,838,680, 6,818,113, 6,614,030, 6,613,525, 6,399,952, and 6,007,690; and US published applications US 2004/0096825, US 2002/0182749, US 2002/0146726, US 2002/0092767, US 2005/0131006, US 2005/0130246, and US 2005/0130238; U.S. Pat. Nos. 6,551,788, 6,867,005, 6,825,041, 6,696,243, 6,740,257, 6,387,672, 6,861,214, 6,843,481, 6,159,749, 6,033,627, 6,387,622, 6,146,833, 5,814,468, 6,110,749, 5,817,468, 6,660,233, 5,882,596, 5,747,352, 5,863,401, 5,571,680, 5,559,012, 5,849,599, 5,447,838, 5,395,754, 5,583,055, 5,371,021, 5,387,527, 5,451,525, 6,168,956, 6,017,767, 5,998,220, 5,877,028, 5,648,274, 5,607,863, 5,468,648, 5,439,829, 5,225,351, 5,296,195, 5,164,311, 5,013,669, 4,788,138, 5,120,504, 5,369,566, 5,139,744, 5,125,748, 5,108,703, 5,104,621, 5,879,881, 4,727,019, 4,595,661, 4,486,530, 4,376,110, and 4,101,276; and US published applications US 2005/0136542, US 2004/0229226, US 2004/0219535, US 2004/0173457, US 2003/0219840, US 2003/0198967, US 2003/0153011, US 2003/0092008, US 2003/0021728, US 2003/0013083, US 2002/0102736, US 2002/0115096, US 2005/0123986, US 2004/0029156, US 2005/0042120, US 2003/0119033, US 2003/0059864, and US 2002/0001853; U.S. Pat. Nos. 5,753,517, 6,509,196 and 6,881,572; and US published applications US 2004/0171092A1, US 2003/0199004A1, US 2003/0071235, and 2003/0162236; U.S. Pat. Nos. 6,341,182, 6,498,010, 6,902,897, 6,897,026, and 6,308,751; and US published applications US 2003/0224447, US 2005/0048672, US 2004/0241700, and US 2003/0071235; US published applications US 2004/0005697, US 2004/0224318, US 2005/0118640, US 2005/0124059, and US 2005/0170356; U.S. Pat. Nos. 6,251,687, 6,830,731, 6,392,894, 6,165,981, 6,121,056, 6,083,708, 5,898,005, 5,861,319, 4,786,606, 4,774,174, 4,752,562, 6,686,208, 6,441,055, 6,440,748, and 6,274,872; and US published application US 2003/0032199; U.S. Pat. Nos. 6,156,270, 6,194,222, 6,391,265, 6,669,907, 6,297,060, and 6,905,882; and U.S. Pat. No. 5,529,902. Those reagents, devices, and system components disclosed in the above-referenced US patents and published patent applications that are suitable for use with the present invention are recognizable by those skilled in the art, and are each hereby incorporated by reference herein.
Results of a multi-analyte assay using the method of the invention are shown in
After benchmarking the basic performance of the assays, the IL-1B antigen concentrations were scaled by adding scaling anti-IL-1B. The response to IL-1B was significantly reduced while the others remained substantially unchanged.
Varied concentrations of five cytokines were spiked into serum and measured using a multiplexed immunoassay. The baseline of the five-plex assay is shown in
In a second set of assays the samples were spiked with 2.3 μg/ml (15.2×10−9 mol/L) of IL-1B scaling antibody to scale the concentration of free IL-1B antigen for detection. As shown in
These data show that multi-analyte assays can be scaled in order to reduce the available concentration of a particular analyte without affecting the response of the other analytes in the system.
This application is based on and claims priority from U.S. Provisional Patent Application Ser. No. 60/650,324, filed Feb. 4, 2005, which is hereby incorporated by reference.
Number | Name | Date | Kind |
---|---|---|---|
4743542 | Graham, Jr. et al. | May 1988 | A |
5028535 | Buechler et al. | Jul 1991 | A |
5089391 | Buechler et al. | Feb 1992 | A |
5356784 | Kauvar | Oct 1994 | A |
5356785 | McMahon et al. | Oct 1994 | A |
5468648 | Chandler | Nov 1995 | A |
5480792 | Buechler et al. | Jan 1996 | A |
5521102 | Boehringer et al. | May 1996 | A |
5541069 | Mortensen et al. | Jul 1996 | A |
5552276 | Mochida et al. | Sep 1996 | A |
5607863 | Chandler | Mar 1997 | A |
5620901 | Kauvar | Apr 1997 | A |
5679526 | Buechler et al. | Oct 1997 | A |
5710005 | Rittenburg | Jan 1998 | A |
5770460 | Pawlak et al. | Jun 1998 | A |
5780308 | Ching et al. | Jul 1998 | A |
5846838 | Chandler | Dec 1998 | A |
5869345 | Chandler | Feb 1999 | A |
5874216 | Mapes | Feb 1999 | A |
5877028 | Chandler et al. | Mar 1999 | A |
5879951 | Sy | Mar 1999 | A |
5939252 | Lennon et al. | Aug 1999 | A |
5939272 | Buechler et al. | Aug 1999 | A |
5962339 | Midgely | Oct 1999 | A |
5968839 | Blatt et al. | Oct 1999 | A |
5985579 | Buechler et al. | Nov 1999 | A |
6027904 | Devine et al. | Feb 2000 | A |
6030770 | Brust | Feb 2000 | A |
6086748 | Durst et al. | Jul 2000 | A |
6136610 | Polito et al. | Oct 2000 | A |
6140134 | Rittenburg | Oct 2000 | A |
6177281 | Manita | Jan 2001 | B1 |
6180340 | Nelson | Jan 2001 | B1 |
6218134 | Yamauchi et al. | Apr 2001 | B1 |
6284472 | Wei et al. | Sep 2001 | B1 |
6316205 | Guan et al. | Nov 2001 | B1 |
6329209 | Wagner et al. | Dec 2001 | B1 |
6410341 | Freitag et al. | Jun 2002 | B1 |
6475805 | Charm et al. | Nov 2002 | B1 |
6498010 | Fitzgerald et al. | Dec 2002 | B1 |
6509196 | Brooks et al. | Jan 2003 | B1 |
6551788 | Bell | Apr 2003 | B1 |
6582970 | Manita | Jun 2003 | B1 |
6617116 | Guan et al. | Sep 2003 | B2 |
6656744 | Pronovost et al. | Dec 2003 | B2 |
6670196 | Buechler | Dec 2003 | B1 |
6680209 | Buechler et al. | Jan 2004 | B1 |
6696243 | Siiman | Feb 2004 | B2 |
20020086337 | Fitzgerald et al. | Jul 2002 | A1 |
20030054571 | Watkins et al. | Mar 2003 | A1 |
20030078737 | Kays et al. | Apr 2003 | A1 |
20050112585 | Zlchl et al. | May 2005 | A1 |
Number | Date | Country |
---|---|---|
2069833 AA | Nov 1992 | CA |
0516095 | Feb 1992 | EP |
0690306 | Mar 1996 | EP |
4351962 | Dec 1992 | JP |
WO 9423298 | Oct 1994 | WO |
WO 9904267 | Jan 1999 | WO |
PCTUS2006003737 | Nov 2006 | WO |
Number | Date | Country | |
---|---|---|---|
20060177873 A1 | Aug 2006 | US |
Number | Date | Country | |
---|---|---|---|
60650324 | Feb 2005 | US |