Patent Publication Number: US-2023160906-A1

Title: Detection of anti-tnf alpha drug biologics and anti-drug antibodies

Description:
CROSS-REFERENCES TO RELATED APPLICATIONS 
     This application is a continuation of PCT/US2020/032859, filed May 14, 2020, which claims priority to U.S. Application No. 62/866,344, filed Jun. 25, 2019, the disclosures of which are hereby incorporated by reference in their entireties for all purposes. 
    
    
     BACKGROUND 
     Tumor necrosis factor alpha (TNFα) is a cytokine produced by numerous cell types, including monocytes and macrophages, that was originally identified based on its ability to induce the necrosis of certain mouse tumors. Subsequently, a factor termed cachectin, associated with cachexia, was shown to be identical to TNFα. TNFα has been implicated in the pathophysiology of a variety of other human diseases and disorders, including shock, sepsis, infections, autoimmune diseases, RA, Crohn&#39;s disease, transplant rejection and graft-versus-host disease. 
     Because of the harmful role of human TNFα (hTNFα) in a variety of human disorders, therapeutic strategies have been designed to inhibit or counteract hTNFα activity. In particular, antibodies that bind to, and neutralize hTNFα, have been sought as a means to inhibit hTNFα activity. Some of the earliest of such antibodies were mouse monoclonal antibodies (mAbs), secreted by hybridomas prepared from lymphocytes of mice immunized with hTNFα (see e.g., U.S. Pat. No. 5,231,024 to Moeller et al.). While these mouse anti-hTNFα antibodies often displayed high affinity for hTNFα and were able to neutralize hTNFα activity, their use in vivo has been limited by problems associated with the administration of mouse antibodies to humans, such as a short serum half-life, an inability to trigger certain human effector functions, and elicitation of an unwanted immune response against the mouse antibody in a human (the “human anti-mouse antibody” (HAMA) reaction). 
     Biological therapies have been applied to the treatment of autoimmune disorders such as rheumatoid arthritis. For example, four TNFα inhibitors, REMICADE™ (infliximab), a chimeric anti-TNFα mAb, ENBREL™ (etanercept), a TNFR-Ig Fc fusion protein, HUMIRA™ (adalimumab), a human anti-TNFα mAb, and CIMZIA® (certolizumab pegol), a PEGylated Fab fragment, have been approved by the FDA for treatment of rheumatoid arthritis. CIMZIA® is also used for the treatment of moderate to severe Crohn&#39;s disease (CD). While such biologic therapies have demonstrated success in the treatment of rheumatoid arthritis and other autoimmune disorders such as CD, not all subjects treated respond, or respond well, to such therapy. Moreover, administration of TNFα inhibitors can induce an immune response to the drug and lead to the production of autoantibodies such as human anti-chimeric antibodies (HACA), human anti-humanized antibodies (HAHA), and human anti-mouse antibodies (HAMA). Such HACA, HAHA, or HAMA immune responses can be associated with hypersensitive reactions and dramatic changes in pharmacokinetics and biodistribution of the immunotherapeutic TNFα inhibitor that preclude further treatment with the drug. 
     In view of the foregoing, there is a need in the art for assays to detect the presence of anti-TNFα biologics and/or their autoantibodies in a patient sample to monitor TNFα inhibitor therapy and to guide treatment decisions. The present disclosure satisfies these needs and provides related advantages as well. 
     BRIEF SUMMARY 
     In one embodiment, the present disclosure provides an assay method for detecting the presence or amount of an anti-TNFα drug in a sample, the method comprising:
         contacting the sample with a TNFα labeled with a first fluorophore;   contacting the sample with an anti-drug antibody or Fab fragment labeled with a second fluorophore;   incubating the sample for a time sufficient to obtain a dual labeled anti-TNFα drug; and   exciting the sample having a dual labeled anti-TNFα drug using a light source to detect a fluorescence emission signal associated with fluorescence resonance energy transfer (FRET).       

     In certain instances, the methods are useful for measuring the levels of REMICADE™ (infliximab), INFLECTRA (Infliximab-dyyb), RENFLEXIS (Infliximab-abda), FLIXABI (Infliximab Biosimilar), REMSIMA (Infliximab Biosimilar), ENBREL™ (etanercept), HUMIRA™ (adalimumab), AMJEVITA (Adalimumab-atto), IMRALDI (Adalimumab Biosimilar), CYLTEZO (Adalimumab Biosimilar), HYRIMOZ (Adalimumab Biosimilar), HULIO (Adalimumab Biosimilar), CIMZIA® (certolizumab pegol), and combinations thereof in a sample, e.g., from a subject receiving such anti-TNFα drug therapy. 
     In another embodiment, the present disclosure provides an assay method for detecting the presence or amount of an anti-TNFα drug in a sample, the method comprising:
         contacting the sample with a TNFα with a first fluorophore;   contacting the sample with an anti-drug Fab fragment labeled with a second fluorophore;   incubating the sample for a time sufficient to obtain a dual labeled anti-TNFα drug; and   exciting the sample have dual labeled anti-TNFα drug using a light source to detect a fluorescence emission signal associated with fluorescence resonance energy transfer (FRET).       

     In yet another aspect, the present disclosure provides an assay method for detecting the presence or amount of an anti-TNFα drug in a sample, the method comprising:
         contacting the sample with a complex comprising an anti-TNFα drug labeled with a first fluorophore and an isolated TNFα labeled with a second fluorophore, wherein the complex emits a fluorescence emission signal associated with fluorescence resonance energy transfer (FRET) when excited using a light source;   incubating the sample with the complex for a time sufficient for anti-TNFα drug in the sample to compete for binding to the anti-TNFα drug labeled with a first fluorophore; and   exciting the sample using a light source to detect a fluorescence emission signal associated with FRET, wherein an absence of the fluorescence emission signal or a decrease in the fluorescence emission signal relative to the fluorescence emission signal initially emitted by the complex, indicates the presence or amount of anti-TNFα drug in the sample.       

     In another embodiment, the present disclosure provides an assay method for detecting the presence or amount of an anti-TNFα drug autoantibody (autoantibody) in a sample, the method comprising:
         contacting the sample with a first labeled anti-TNFα drug or Fab fragment with a donor fluorophore;   contacting the sample with a second labeled anti-TNFα drug or Fab fragment with an acceptor fluorophore;   incubating the sample for a time sufficient to generate a ternary complex of the first labeled anti-TNFα drug with a donor fluorophore, the second labeled anti-TNFα drug or Fab fragment labeled with an acceptor fluorophore and the autoantibody; and   exciting the sample having the ternary complex using a light source to detect a fluorescence emission signal associated with fluorescence resonance energy transfer (FRET) when the donor fluorophore is excited.       

     These and other aspects, objects and embodiments will become more apparent when read with the detailed description and figures that follow. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG.  1 A  illustrates one embodiment of the present disclosure for detecting anti-TNFα drug concentration. 
         FIG.  1 B  illustrates one embodiment of the present disclosure for detecting anti-TNFα drug concentration. 
         FIG.  2    illustrates a standard curve generated using methods of the present disclosure. 
         FIG.  3    illustrates one embodiment of a donor fluorophore of the present disclosure. 
         FIG.  4    illustrates one embodiment of an acceptor fluorophore of the present disclosure. 
         FIG.  5    illustrates donor and acceptor wavelengths in one embodiment of the present disclosure. Tb-H22TRENIAM-5LIO-NHS emission profile is shown (490 nm, 545 nm, 580 nm and 620 nm). Acceptor emission peaks are shown in (AF488, second arrow from left), (AF546, fourth arrow from left) and (AF647, seventh arrow from the left i.e., first arrow on the right). 
         FIG.  6    illustrates a trFRET adalimumab (ADA) assay which reaches equilibrium after 3 minutes. Eight concentrations ranging from 0 μg/mL to 50 μg/m were tested. Reagents were mixed with a sample and read at different time points as shown. 
         FIG.  7 A  illustrates one embodiment of the present disclosure for detecting anti-drug antibody (autoantibodies). 
         FIG.  7 B  illustrates one embodiment of the present disclosure for detecting anti-drug antibody (autoantibodies). 
         FIG.  8    illustrates a standard curve generated using methods of the present disclosure. 
         FIG.  9    illustrates a comparison of methods of the present disclosure and commercially available method. 
         FIG.  10    illustrates a standard curve generated using methods of the present disclosure. 
         FIG.  11    illustrates a comparison of methods of the present disclosure and commercially available method. 
     
    
    
     DETAILED DESCRIPTION 
     I. Definitions 
     The terms “a,” “an,” or “the” as used herein not only includes aspects with one member, but also includes aspects with more than one member. 
     The term “about” as used herein to modify a numerical value indicates a defined range around that value. If “X” were the value, “about X” would indicate a value from 0.9X to 1.1X, and more preferably, a value from 0.95X to 1.05X. Any reference to “about X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, “about X” is intended to teach and provide written description support for a claim limitation of, e.g., “0.98X.” 
     When the modifier “about” is applied to describe the beginning of a numerical range, it applies to both ends of the range. Thus, “from about 500 to 850 nm” is equivalent to “from about 500 nm to about 850 nm.” When “about” is applied to describe the first value of a set of values, it applies to all values in that set. Thus, “about 580, 700, or 850 nm” is equivalent to “about 580 nm, about 700 nm, or about 850 nm.” 
     “Activated acyl” as used herein includes a —C(O)-LG group. “Leaving group” or “LG” is a group that is susceptible to displacement by a nucleophilic acyl substitution (i.e., a nucleophilic addition to the carbonyl of —C(O)-LG, followed by elimination of the leaving group). Representative leaving groups include halo, cyano, azido, carboxylic acid derivatives such as t-butylcarboxy, and carbonate derivatives such as i-BuOC(O)O—. An activated acyl group may also be an activated ester as defined herein or a carboxylic acid activated by a carbodiimide to form an anhydride (preferentially cyclic) or mixed anhydride —OC(O)Ra or —OC(NR a )NHR b  (preferably cyclic), wherein Ra and Rb are members independently selected from the group consisting of C 1 -C 6  alkyl, C 1 -C 6  perfluoroalkyl, C 1 -C 6  alkoxy, cyclohexyl, 3-dimethylaminopropyl, or N-morpholinoethyl. Preferred activated acyl groups include activated esters. 
     “Activated ester” as used herein includes a derivative of a carboxyl group that is more susceptible to displacement by nucleophilic addition and elimination than an ethyl ester group (e.g., an NHS ester, a sulfo-NHS ester, a PAM ester, or a halophenyl ester). Representative carbonyl substituents of activated esters include succinimidyloxy (—OC 4 H 4 NO 2 ), sulfosuccinimidyloxy (—OC 4 H 3 NO 2 SO 3 H), -1-oxybenzotriazolyl (—OC 6 H 4 N 3 ); 4-sulfo-2,3,5,6-tetrafluorophenyl; or an aryloxy group that is optionally substituted one or more times by electron-withdrawing substituents such as nitro, fluoro, chloro, cyano, trifluoromethyl, or combinations thereof (e.g., pentafluorophenyloxy, or 2,3,5,6-tetrafluorophenyloxy). Preferred activated esters include succinimidyloxy, sulfosuccinimidyloxy, and 2,3,5,6-tetrafluorophenyloxy esters. 
     “FRET partners” refer to a pair of fluorophores consisting of a donor fluorescent compound such as cryptate and an acceptor compound such as Alexa 647, when they are in proximity to one another and when they are excited at the excitation wavelength of the donor fluorescent compound, these compounds emit a FRET signal. It is known that, in order for two fluorescent compounds to be FRET partners, the emission spectrum of the donor fluorescent compound must at least partially overlap the excitation spectrum of the acceptor compound. The preferred FRET-partner pairs are those for which the value R0 (Förster distance, distance at which energy transfer is 50% efficient) is less than or equal to 100 Å. 
     “Fluorescence resonance energy transfer (FRET)” or “Förster resonance energy transfer (FRET)” refer to a mechanism describing energy transfer between a donor compound such as a lanthanide cryptate and an acceptor compound such as Alexa 647, when they are in proximity to one another and when they are excited at the excitation wavelength of the donor fluorescent compound. A donor compound, initially in its electronic excited state, may transfer energy to an acceptor fluorophore through nonradiative dipole-dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance. 
     “FRET signal” refers to any measurable signal representative of FRET between a donor fluorescent compound and an acceptor compound. A FRET signal can therefore be a variation in the intensity or in the lifetime of luminescence of the donor fluorescent compound or of the acceptor compound when the latter is fluorescent. 
     The terms “anti-TNFα drug” or “TNFα inhibitor” as used herein is intended to encompass agents including proteins, antibodies, antibody fragments, fusion proteins (e.g., Ig fusion proteins or Fc fusion proteins), multivalent binding proteins (e.g., DVD Ig), small molecule TNFα antagonists and similar naturally or non-naturally-occurring molecules, and/or recombinant and/or engineered forms thereof, that, directly or indirectly, inhibit TNF a activity, such as by inhibiting interaction of TNFα with a cell surface receptor for TNFα, inhibiting TNFα protein production, inhibiting TNFα gene expression, inhibiting TNFα secretion from cells, inhibiting TNFα receptor signaling or any other means resulting in decreased TNFα activity in a subject. The term “anti-TNFα drug” or “TNFα inhibitor” preferably includes agents which interfere with TNFα activity. Examples of TNFα inhibitors include etanercept (ENBREL™, Amgen), infliximab (REMICADE™, Johnson and Johnson), human anti-TNF monoclonal antibody adalimumab (D2E7/HUMIRA™, Abbott Laboratories), certolizumab pegol (CIMZIA®, UCB, Inc.), CDP 571 (Celltech), and CDP 870 (Celltech), as well as other compounds which inhibit TNF a activity, such that when administered to a subject suffering from or at risk of suffering from a disorder in which TNF a activity is detrimental (e.g., RA), the disorder is treated. 
     The term “anti-drug antibody” refers to an antibody of an anti-TNFα drug or a TNFα inhibitor. Examples include an antibody to TNFα inhibitors such as etanercept (ENBREL™, Amgen), infliximab (REMICADE′, Johnson and Johnson), human anti-TNF monoclonal antibody adalimumab (D2E7/HUMIRA™, Abbott Laboratories), certolizumab pegol (CIMZIA®, UCB, Inc.), CDP 571 (Celltech), and CDP 870 (Celltech). In most instances, anti-TNFα drugs are themselves antibodies. Thus, an “anti-drug antibody” is an antibody of an antibody. Bio-Rad product codes for anti-infliximab antibodies include HCA214, HCA215, HCA216, and HCA216P. 
     In certain embodiments, “TNFα” is an “antigen,” which is a molecule or a portion of the molecule capable of being bound by an anti-TNFα antibody. In certain instances, TNFα will react, in a highly selective manner, with an anti-TNFα antibody. In certain instances, TNFα is a sufficient length having an epitope of TNF a that is capable of binding anti-TNFα antibodies, fragments and regions thereof. 
     I. Embodiments 
     The importance of measuring serum concentrations of anti-TNFα biologics as well as other immunotherapeutics is illustrated by the fact that the FDA requires pharmacokinetic and tolerability (e.g., immune response) studies to be performed during clinical trials. The present disclosure finds utility in monitoring patients receiving these drugs to make sure they are getting the right dose, that the drug is not being cleared from the body too quickly, and that the patient is not developing an immune response against the drug. Furthermore, the present disclosure is useful in guiding the switch between different drugs due to failure with the initial drug. 
     In one embodiment, the present disclosure provides an assay method for detecting the presence or amount of an anti-TNFα drug in a sample, the method comprising:
         contacting the sample with a TNFα labeled with a first fluorophore;   contacting the sample with an anti-drug antibody or Fab fragment labeled with a second fluorophore;   incubating the sample for a time sufficient to obtain a dual labeled anti-TNFα drug; and   exciting the sample having a dual labeled anti-TNFα drug using a light source to detect fluorescence emission signal associated with fluorescence resonance energy transfer (FRET).       

     In another embodiment, the present disclosure provides an assay method for detecting the presence or amount of an anti-TNFα drug in a sample, the method comprising:
         contacting the sample with a TNFα with a first fluorophore;   contacting the sample with anti-drug Fab fragment labeled with a second fluorophore;   incubating the sample for a time sufficient to obtain a dual labeled anti-TNFα drug; and   exciting the sample have dual labeled anti-TNFα drug using a light source to detect fluorescence emission signal associated with fluorescence resonance energy transfer (FRET).       

     In yet another aspect, the present disclosure provides an assay method for detecting the presence or amount of an anti-TNFα drug in a sample, the method comprising:
         contacting the sample with a complex comprising an anti-TNFα drug labeled with a first fluorophore and an isolated TNFα labeled with a second fluorophore, wherein the complex emits a fluorescence emission signal associated with fluorescence resonance energy transfer (FRET) when excited using a light source;   incubating the sample with the complex for a time sufficient for anti-TNFα drug in the sample to compete for binding to the anti-TNFα drug labeled with a first fluorophore; and
 
exciting the sample using a light source to detect a fluorescence emission signal associated with FRET, wherein an absence of the fluorescence emission signal or a decrease in the fluorescence emission signal relative to the fluorescence emission signal initially emitted by the complex indicates the presence or amount of anti-TNFα drug in the sample.
       

     In the foregoing embodiment, anti-TNFα drug in the sample competes with the anti-TNFα drug labeled with a first fluorophore. The greater the decrease in the fluorescence emission signal relative to the fluorescence emission signal initially emitted by the complex indicates a greater amount of anti-TNFα drug in the sample 
     In certain instances, the methods are useful for measuring or monitoring the presence, level or concentration of biologics such as REMICADE™ (infliximab), INFLECTRA (Infliximab-dyyb), RENFLEXIS (Infliximab-abda), FLIXABI (Infliximab Biosimilar), REMSIMA (Infliximab Biosimilar), ENBREL™ (etanercept), HUMIRA™ (adalimumab), AMJEVITA (Adalimumab-atto), IMRALDI (Adalimumab Biosimilar), CYLTEZO (Adalimumab Biosimilar), HYRIMOZ (Adalimumab Biosimilar), HULIO (Adalimumab Biosimilar), CIMZIA® (certolizumab pegol), and combinations thereof in a sample, e.g., from a subject receiving such anti-TNFα drug therapy. 
     In certain aspects, the FRET assay is a time-resolved FRET assay. The fluorescence emission signal or measured FRET signal is directly correlated with the biological phenomenon studied. In fact, the level of energy transfer between the donor compound and the acceptor fluorescent compound is proportional to the reciprocal of the distance between these compounds to the 6 th  power. For the donor/acceptor pairs commonly used by those skilled in the art, the distance Ro (corresponding to a transfer efficiency of 50%) is in the order of 1, 5, 10, 20 or 30 nanometers. 
     In certain aspects, the sample is a biological sample. Suitable biological samples include, but are not limited to, whole blood, plasma, serum, blood cells, cell samples, urine, spinal fluid, sweat, tear fluid, saliva, skin, mucous membrane, and hair. As a sample, whole blood, plasma, serum, blood cells and such are preferred, and whole blood, blood cells, and such are particularly preferred. Whole blood includes samples of whole blood-derived blood cell fractions admixed with plasma. With regard to these samples, samples subjected to pretreatments such as hemolysis, separation, dilution, concentration, and purification can be used. In a one aspect, the biological sample is a whole blood or a serum sample. 
     In certain aspects, the first fluorophore is a donor and the second fluorophore is an acceptor. 
     In certain aspects, the first fluorophore is an acceptor and the second fluorophore is a donor. 
     In certain aspects, the FRET energy donor compound is a cryptate, such as a lanthanide cryptate. 
     In certain aspects, as shown in  FIG.  1 A , the FRET assay format for the anti-TNFα drug is a FRET anti-antibody bridge assay. The assay includes contacting the sample with a TNFα labeled with a first fluorophore, such as a donor fluorophore. The sample is also contacted with an anti-drug antibody or Fab fragment labeled with a second fluorophore, such as an acceptor fluorophore. These contacting steps can be simultaneous or sequential. The sample incubates for a time sufficient to obtain a dual labeled anti-TNFα drug. The sample is then excited using a light source to detect a fluorescence emission signal associated with fluorescence resonance energy transfer (FRET). An increasing FRET signal indicates increased concentration of the anti-TNFα drug. 
     In certain aspects, as shown in  FIG.  1 B , the FRET assay format, for example, of adalimumab (e.g., DRUG, an anti-TNFα drug) is an Amplified Sandwich TR-FRET assay. The TNFα labeled with a donor fluorophore binds to the anti-TNFα drug (e.g. adalimumab). The anti-adalimumab Fab with an acceptor fluorophore preferably binds to the adalimumab Fab region when TNFα is also bound. In one aspect, the anti-adalimumab Fab with an acceptor fluorophore binds to the adalimumab Fab region only when TNFα is also bound. Each analyte (anti-TNFα drug, e.g., adalimumab) contains two Fab regions, enabling multiple bindings of TNFα donor and anti-adalimumab Fab acceptor, thus yielding twice the possible FRET signal per analyte. In this aspect, one or more TNFα labeled donors can bind and one or more anti-Fab acceptors can bind. 
     In addition, in  FIG.  1 B , each Fab region on the analyte (anti-TNFα drug, e.g., adalimumab) can contain the TNFα donor and anti-adalimumab Fab acceptor in close proximity, enabling energy transfer producing an “amplified” or increased FRET signal. An increasing FRET signal indicates increased concentration of the anti-TNFα drug. 
     In certain aspects, the cryptate has an absorption wavelength between about 300 nm to about 400 nm. In certain aspects, as shown in  FIG.  4   , cryptate dyes have four fluorescence emission peaks at about 490 nm, about 545 nm, about 580 nm, and 620 nm. Thus, as a donor, the cryptate is compatible with fluorescein-like (green zone) molecules, Cy5, DY-647-like (red zone) acceptors, Allophycocyanin (APC), or Phycoeruythrin (PE) to perform TR-FRET experiments. 
     In certain aspects of the embodiments, the introduction of a time delay between a flash excitation and the measurement of the fluorescence at the acceptor emission wavelength allows to discriminate long lived from short-lived fluorescence and to increase signal-to-noise ratio. 
     In certain aspects of the embodiments, the detection device detects time-resolved (tr) fluorescent signal from both the donor and FRET acceptor emission. Time-resolved (tr) FRET is a technique to improve signal to noise by removing short-lived fluorescent signals originating from the sample. The donor fluorophore is excited using a pulse of light. The emission from both the donor and acceptor signals are read after a time delay from the end of the excitation pulse. Noise is reduced as background fluorescence from nonspecific sources decay more rapidly than the emitted light from the donor allowing the acceptor signal to be read long after the nonspecific fluorescence has passed. 
     In certain aspects of the embodiments, the assay uses a donor fluorophore consisting of terbium bound within a cryptate. The terbium cryptate can be excited with a 365 nm UV LED. The terbium cryptate emits at four (4) wavelengths within the visible region. In one aspect, the assay uses the lowest donor emission energy peak of 620 nm as the donor signal within the assay. In certain aspects, the acceptor fluorophore, when in very close proximity, is excited by the highest energy terbium cryptate emission peak of 490 nm causing light emission at 520 nm. Both the 620 nm and 520 nm emission wavelengths are measured independently in a device or instrument and results can be reported as RFU ratio 620/520. 
     In certain instances, the methods are useful for measuring the levels of autoantibodies including, but not limited to, human anti-chimeric antibodies (HACA), human anti-humanized antibodies (HAHA), and human anti-mouse antibodies (HAMA) in a sample, e.g., from a subject receiving anti-TNFα drug therapy. The autoantibodies can be non-neutralizing or neutralizing autoantibodies. 
     As such, the present disclosure provides an assay method for detecting the presence or amount of an anti-TNFα drug autoantibody (autoantibody) in a sample, the method comprising:
         contacting the sample with a first labeled anti-TNFα drug or Fab fragment with a donor fluorophore;   contacting the sample with a second labeled anti-TNFα drug or Fab fragment with an acceptor fluorophore;   incubating the sample for a time sufficient to generate a ternary complex of the first labeled anti-TNFα drug with a donor fluorophore, the second labeled anti-TNFα drug or Fab fragment labeled with an acceptor fluorophore and the autoantibody; and   exciting the sample having the ternary complex using a light source to detect a fluorescence emission signal associated with fluorescence resonance energy transfer (FRET) when the donor fluorophore is excited.       

     1. Cryptates as FRET Donors 
     In certain aspects, the terbium cryptate molecule “Lumi4-Tb” from Lumiphore, marketed by Cisbio bioassays is used as the cryptate donor. The terbium cryptate “Lumi4-Tb” having the formula below, which can be coupled to an antibody by a reactive group, in this case, for example, an NHS ester: 
     
       
         
         
             
             
         
       
     
     An activated ester (an NHS ester) can react with a primary amine on an antibody to make a stable amide bond. A maleimide on the cryptate and a thiol on the antibody can react together and make a thioether. Alkyl halides react with amines and thiols to make alkylamines and thioethers, respectively. Any derivative providing a reactive moiety that can be conjugated to an antibody can be utilized herein. 
     In certain other aspects, cryptates disclosed in WO2015157057, titled “Macrocycles” are suitable for use in the present disclosure. This publication contains cryptate molecules useful for labeling biomolecules. As disclosed therein, certain of the cryptates have the structure as follows: 
     
       
         
         
             
             
         
       
     
     In certain other aspects, a terbium cryptate useful in the present disclosure is shown below: 
     
       
         
         
             
             
         
       
     
     In certain aspects, the cryptates that are useful in the present invention are disclosed in WO 2018/130988, published Jul. 19, 2018. As disclosed therein, the compounds of Formula I are useful as FRET donors in the present disclosure: 
     
       
         
         
             
             
         
       
         
         
           
             wherein when the dotted line is present, R and R 1  are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, alkyl optionally substituted with one or more halogen atoms, carboxyl, alkoxycarbonyl, amido, sulfonato, alkoxycarbonylalkyl or alkylcarbonylalkoxy or alternatively, R and R 1  join to form an optionally substituted cyclopropyl group wherein the dotted bond is absent; 
             R 2  and R 3  are each independently a member selected from the group consisting of hydrogen, halogen, SO 3 H, —SO 2 —X, wherein X is a halogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, or an activated group that can be linked to a biomolecule, wherein the activated group is a member selected from the group consisting of a halogen, an activated ester, an activated acyl, optionally substituted alkylsulfonate ester, optionally substituted arylsulfonate ester, amino, formyl, glycidyl, halo, haloacetamidyl, haloalkyl, hydrazinyl, imido ester, isocyanato, isothiocyanato, maleimidyl, mercapto, alkynyl, hydroxyl, alkoxy, amino, cyano, carboxyl, alkoxycarbonyl, amido, sulfonato, alkoxycarbonylalkyl, cyclic anhydride, alkoxyalkyl, a water solubilizing group or L; 
             R 4  are each independently a hydrogen, C 1 -C 6  alkyl, or alternatively, 3 of the R 4  groups are absent and the resulting oxides are chelated to a lanthanide cation; and 
             Q 1 -Q 4  are each independently a member selected from the group of carbon or nitrogen. 
           
         
       
    
     2. FRET Acceptors 
     In order to detect a FRET signal, a FRET acceptor is required. The FRET acceptor has an excitation wavelength that overlaps with an emission wavelength of the FRET donor. The FRET signal of the acceptor is proportional to the concentration level of analyte present in the sample, such as a patient&#39;s blood sample as interpolated from a known amount of calibrators i.e., a standard curve. 
     The acceptor molecules that can be used include, but are not limited to, fluorescein-like (green zone) acceptor, Cy5, DY-647, Alexa Fluor 488, Alexa Fluor 546, Allophycocyanin (APC), Phycoeruythrin (PE) and Alexa Fluor 647. Donor and acceptor fluorophores having reactive moieties such as an NHS ester can be conjugated using a primary amine on an antibody. 
     Other acceptors include, but are not limited to, cyanine derivatives, D2, CYS, fluorescein, coumarin, rhodamine, carbopyronine, oxazine and its analogs, Alexa Fluor fluorophores, Crystal violet, perylene bisimide fluorophores, squaraine fluorophores, boron dipyrromethene derivatives, NBD (nitrobenzoxadiazole) and its derivatives, DABCYL (4-((4-(dimethylamino)phenyl)azo)benzoic acid). 
     In one aspect, fluorescence can be characterized by wavelength, intensity, lifetime, polarization or a combination thereof 
     3. Antibodies 
     In certain aspects, an activated ester (an NHS ester) of the donor or acceptor can react with a primary amine on an antibody to make a stable amide bond. For example, a maleimide on the cryptate or the acceptor (e.g., Alexa 647) and a thiol on the antibody can react together and make a thioether. Alkyl halides react with amines and thiols to make alkylamines and thioethers, respectively. Any derivative providing a reactive moiety that can be conjugated to an antibody can be utilized herein to make the first antibody labeled with a donor fluorophore specific for the analyte, as well as, the second antibody labeled with an acceptor fluorophore specific for analyte. 
     The methods herein can use a variety of samples, which include a tissue sample, blood, biopsy, serum, plasma, saliva, urine, or stool sample. 
     4. Production of Antibodies 
     The generation and selection of antibodies not already commercially available can be accomplished several ways. For example, one way is to express and/or purify a polypeptide of interest (i.e., antigen) using protein expression and purification methods known in the art, while another way is to synthesize the polypeptide of interest using solid phase peptide synthesis methods known in the art. See, e.g.,  Guide to Protein Purification , Murray P. Deutcher, ed.,  Meth. Enzymol ., Vol. 182 (1990);  Solid Phase Peptide Synthesis , Greg B. Fields, ed.,  Meth. Enzymol., Vol.  289 (1997); Kiso et al.,  Chem. Pharm. Bull.,  38:1192-99 (1990); Mostafavi et al.,  Biomed. Pept. Proteins Nucleic Acids,  1:255-60, (1995); and Fujiwara et al.,  Chem. Pharm. Bull.,  44:1326-31 (1996). The purified or synthesized polypeptide can then be injected, for example, into mice or rabbits, to generate polyclonal or monoclonal antibodies. One skilled in the art will recognize that many procedures are available for the production of antibodies, for example, as described in  Antibodies, A Laboratory Manual , Harlow and Lane, Eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988). One skilled in the art will also appreciate that binding fragments or Fab fragments which mimic antibodies can also be prepared from genetic information by various procedures (see, e.g.,  Antibody Engineering: A Practical Approach , Borrebaeck, Ed., Oxford University Press, Oxford (1995); and Huse et al.,  J. Immunol.,  149:3914-3920 (1992)). 
     In addition, numerous publications have reported the use of phage display technology to produce and screen libraries of polypeptides for binding to a selected target antigen (see, e.g, Cwirla et al.,  Proc. Natl. Acad. Sci. USA,  87:6378-6382 (1990); Devlin et al.,  Science,  249:404-406 (1990); Scott et al.,  Science,  249:386-388 (1990); and Ladner et al., U.S. Pat. No. 5,571,698). A basic concept of phage display methods is the establishment of a physical association between a polypeptide encoded by the phage DNA and a target antigen. This physical association is provided by the phage particle, which displays a polypeptide as part of a capsid enclosing the phage genome which encodes the polypeptide. The establishment of a physical association between polypeptides and their genetic material allows simultaneous mass screening of very large numbers of phage bearing different polypeptides. Phage displaying a polypeptide with affinity to a target antigen bind to the target antigen and these phage are enriched by affinity screening to the target antigen. The identity of polypeptides displayed from these phage can be determined from their respective genomes. Using these methods, a polypeptide identified as having a binding affinity for a desired target antigen can then be synthesized in bulk by conventional means (see, e.g., U.S. Pat. No. 6,057,098). 
     The antibodies that are generated by these methods can then be selected by first screening for affinity and specificity with the purified polypeptide antigen of interest and, if required, comparing the results to the affinity and specificity of the antibodies with other polypeptide antigens that are desired to be excluded from binding. The screening procedure can involve immobilization of the purified polypeptide antigens in separate wells of microtiter plates. The solution containing a potential antibody or group of antibodies is then placed into the respective microtiter wells and incubated for about 30 minutes to 2 hours. The microtiter wells are then washed and a labeled secondary antibody (e.g., an anti-mouse antibody conjugated to alkaline phosphatase if the raised antibodies are mouse antibodies) is added to the wells and incubated for about 30 minutes and then washed. Substrate is added to the wells and a color reaction will appear where antibody to the immobilized polypeptide antigen is present. 
     The antibodies so identified can then be further analyzed for affinity and specificity. In the development of immunoassays for a target protein (glycated hemoglobin), the purified target protein acts as a standard with which to judge the sensitivity and specificity of the immunoassay using the antibodies that have been selected. Because the binding affinity of various antibodies may differ, e.g., certain antibody combinations may interfere with one another sterically, assay performance of an antibody may be a more important measure than absolute affinity and specificity of that antibody. 
     Those skilled in the art will recognize that many approaches can be taken in producing antibodies or binding fragments and screening and selecting for affinity and specificity for the various polypeptides of interest, but these approaches do not change the scope of the present invention. 
     A. Polyclonal Antibodies 
     Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of a polypeptide of interest and an adjuvant. It may be useful to conjugate the polypeptide of interest to a protein carrier that is immunogenic in the species to be immunized, such as, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent. Non-limiting examples of bifunctional or derivatizing agents include maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (conjugation through lysine residues), glutaraldehyde, succinic anhydride, SOCl 2 , and R 1 N═C═NR, wherein R and R 1  are different alkyl groups. 
     Animals are immunized against the polypeptide of interest or an immunogenic conjugate or derivative thereof by combining, e.g., 100 μg (for rabbits) or 5 μg (for mice) of the antigen or conjugate with 3 volumes of Freund&#39;s complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with about ⅕ to 1/10 the original amount of polypeptide or conjugate in Freund&#39;s incomplete adjuvant by subcutaneous injection at multiple sites. Seven to fourteen days later, the animals are bled and the serum is assayed for antibody titer. Animals are typically boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same polypeptide, but conjugation to a different immunogenic protein and/or through a different cross-linking reagent may be used. Conjugates can also be made in recombinant cell culture as fusion proteins. In certain instances, aggregating agents such as alum can be used to enhance the immune response. 
     B. Monoclonal Antibodies 
     Monoclonal antibodies are generally obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. For example, monoclonal antibodies can be made using the hybridoma method described by Kohler et al.,  Nature,  256:495 (1975) or by any recombinant DNA method known in the art (see, e.g., U.S. Pat. No. 4,816,567). 
     In the hybridoma method, a mouse or other appropriate host animal (e.g., hamster) is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies which specifically bind to the polypeptide of interest used for immunization. Alternatively, lymphocytes are immunized in vitro. The immunized lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (see, e.g., Goding,  Monoclonal Antibodies: Principles and Practice , Academic Press, pp. 59-103 (1986)). The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances which inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT), the culture medium for the hybridoma cells will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells. 
     Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and/or are sensitive to a medium such as HAT medium. Examples of such preferred myeloma cell lines for the production of human monoclonal antibodies include, but are not limited to, murine myeloma lines such as those derived from MOPC-21 and MPC-11 mouse tumors (available from the Salk Institute Cell Distribution Center; San Diego, Calif.), SP-2 or X63-Ag8-653 cells (available from the American Type Culture Collection; Rockville, Md.), and human myeloma or mouse-human heteromyeloma cell lines (see, e.g., Kozbor,  J. Immunol.,  133:3001 (1984); and Brodeur et al.,  Monoclonal Antibody Production Techniques and Applications , Marcel Dekker, Inc., New York, pp. 51-63 (1987)). 
     The culture medium in which hybridoma cells are growing can be assayed for the production of monoclonal antibodies directed against the polypeptide of interest. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as a radioimmunoassay (RIA) or an enzyme-linked immunoabsorbent assay (ELISA). The binding affinity of monoclonal antibodies can be determined using, e.g., the Scatchard analysis of Munson et al.,  Anal. Biochem.,  107:220 (1980). 
     After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (see, e.g., Goding,  Monoclonal Antibodies: Principles and Practice , Academic Press, pp. 59-103 (1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal. The monoclonal antibodies secreted by the subclones can be separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. 
     DNA encoding the monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as  E. coli  cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody, to induce the synthesis of monoclonal antibodies in the recombinant host cells. See, e.g., Skerra et al.,  Curr. Opin. Immunol.,  5:256-262 (1993); and Pluckthun, Immunol Rev., 130:151-188 (1992). The DNA can also be modified, for example, by substituting the coding sequence for human heavy chain and light chain constant domains in place of the homologous murine sequences (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al.,  Proc. Natl. Acad. Sci. USA,  81:6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. 
     In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in, for example, McCafferty et al.,  Nature,  348:552-554 (1990); Clackson et al.,  Nature,  352:624-628 (1991); and Marks et al.,  J. Mol. Biol.,  222:581-597 (1991). The production of high affinity (nM range) human monoclonal antibodies by chain shuffling is described in Marks et al.,  BioTechnology,  10:779-783 (1992). The use of combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries is described in Waterhouse et al.,  Nuc. Acids Res.,  21:2265-2266 (1993). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma methods for the generation of monoclonal antibodies. Human Antibodies 
     As an alternative to humanization, human antibodies can be generated. In some embodiments, transgenic animals (e.g., mice) can be produced that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al.,  Proc. Natl. Acad. Sci. USA,  90:2551 (1993); Jakobovits et al.,  Nature,  362:255-258 (1993); Bruggermann et al.,  Year in Immun.,  7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369, and 5,545,807. 
     Alternatively, phage display technology (see, e.g., McCafferty et al.,  Nature,  348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro, using immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats as described in, e.g., Johnson et al.,  Curr. Opin. Struct. Biol.,  3:564-571 (1993). Several sources of V-gene segments can be used for phage display. See, e.g., Clackson et al.,  Nature,  352:624-628 (1991). A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described in Marks et al.,  J. Mol. Biol.,  222:581-597 (1991); Griffith et al.,  EHBO J.,  12:725-734 (1993); and U.S. Pat. Nos. 5,565,332 and 5,573,905. 
     In certain instances, human antibodies can be generated by in vitro activated B cells as described in, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275. 
     C. Antibody Fragments 
     Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,  J. Biochem. Biophys. Meth.,  24:107-117 (1992); and Brennan et al.,  Science,  229:81 (1985)). However, these fragments can now be produced directly using recombinant host cells. For example, the antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab′-SH fragments can be directly recovered from  E. coli  cells and chemically coupled to form F(ab′)2 fragments (see, e.g., Carter et al.,  BioTechnology,  10:163-167 (1992)). According to another approach, F(ab′)2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See, e.g., PCT Publication No. WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. The antibody fragment may also be a linear antibody as described, e.g., in U.S. Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific. 
     D. Bispecific Antibodies 
     Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the same polypeptide of interest. Other bispecific antibodies may combine a binding site for the polypeptide of interest with binding site(s) for one or more additional antigens. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab′)2 bispecific antibodies). 
     Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (see, e.g., Millstein et al.,  Nature,  305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually performed by affinity chromatography. Similar procedures are disclosed in PCT Publication No. WO 93/08829 and Traunecker et al.,  EMBO J.,  10:3655-3659 (1991). 
     According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy chain constant region (CH1) containing the site necessary for light chain binding present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance. 
     In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. See, e.g., PCT Publication No. WO 94/04690 and Suresh et al.,  Meth. Enzymol.,  121:210 (1986). 
     According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side-chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side-chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side-chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. 
     Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable cross-linking agents and techniques are well-known in the art, and are disclosed in, e.g., U.S. Pat. No. 4,676,980. 
     Suitable techniques for generating bispecific antibodies from antibody fragments are also known in the art. For example, bispecific antibodies can be prepared using chemical linkage. In certain instances, bispecific antibodies can be generated by a procedure in which intact antibodies are proteolytically cleaved to generate F(ab′)2 fragments (see, e.g., Brennan et al.,  Science,  229:81 (1985)). These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody. 
     In some embodiments, Fab′-SH fragments can be directly recovered from  E. coli  and chemically coupled to form bispecific antibodies. For example, a fully humanized bispecific antibody F(ab′)2 molecule can be produced by the methods described in Shalaby et al.,  J. Exp. Med.,  175: 217-225 (1992). Each Fab′ fragment was separately secreted from  E. coli  and subjected to directed chemical coupling in vitro to form the bispecific antibody. 
     Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. See, e.g., Kostelny et al.,  J. Immunol.,  148:1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al.,  Proc. Natl. Acad. Sci. USA,  90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers is described in Gruber et al.,  J. Immunol.,  152:5368 (1994). 
     Antibodies with more than two valencies are also contemplated. For example, trispecific antibodies can be prepared. See, e.g., Tutt et al.,  J. Immunol.,  147:60 (1991). 
     E. Antibody Purification 
     When using recombinant techniques, antibodies can be produced inside an isolated host cell, in the periplasmic space of a host cell, or directly secreted from a host cell into the medium. If the antibody is produced intracellularly, the particulate debris is first removed, for example, by centrifugation or ultrafiltration. Carter et al.,  BioTech.,  10:163-167 (1992) describes a procedure for isolating antibodies which are secreted into the periplasmic space of  E. coli . Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) for about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants. 
     The antibody composition prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γ1, γ2, or γ4 heavy chains (see, e.g., Lindmark et al.,  J. Immunol. Meth.,  62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (see, e.g., Guss et al.,  EMBO J.,  5:1567-1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker; Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered. 
     Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25 M salt). 
     In certain aspects, the methods are useful for detecting the amount of anti-TNFα drugs such as, e.g., antibodies including REMICADE™ (infliximab), INFLECTRA (Infliximab-dyyb), RENFLEXIS (Infliximab-abda), FLIXABI (Infliximab Biosimilar), REMSIMA (Infliximab Biosimilar), ENBREL™ (etanercept), HUMIRA™ (adalimumab), AMJEVITA (Adalimumab-alto), IMRALDI (Adalimumab Biosimilar), CYLTEZO (Adalimumab Biosimilar), HYRIMOZ (Adalimumab Biosimilar), HULIO (Adalimumab Biosimilar), and CIMZIA® (certolizumab pegol). 
     In certain aspects, the phrase “high level of an anti-TNFα drug” includes drug levels of about 10 to about 100 ng/10 μL, about 10 to about 70 ng/10 μL, or about 10 to about 50 ng/10 μL. In other embodiments, the phrase “high level of anti-TNFα drug” includes drug levels greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ng/10 μL. 
     In certain aspects, the phrase “medium level of an anti-TNFα drug” includes drug levels of about 5.0 to about 50 ng/10 μL, about 5.0 to about 30 ng/10 μL, about 5.0 to about 20 ng/10 μL, or about 5.0 to about 10 ng/10 μL. In other embodiments, the phrase “medium level of anti-TNFα drug” includes drug levels of about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 ng/10 μL. 
     In certain aspects, the phrase “low level of an anti-TNFα drug” includes drug levels of about 0 to about 10 ng/10 μl, about 0 to about 8 ng/10 μL, or about 0 to about 5 ng/10 μl. In other embodiments, the phrase “low level of an anti-TNFα drug” includes drug levels of about less than about 10, 9.0, 8.0, 7.0, 6.0, 5.0, 4.0, 3.0, 2.0, 1.0, or 0.5 ng/10 μl 
     In certain aspects, the acronym “ADA” includes the phrase “anti-drug antibody.” These or autoantibodies are an autoimmune reaction to the use of biologics. In other aspects, “ADA” is short hand for “Adalimumab.” 
     In certain aspects, the phrase “high level of an anti-drug antibody” includes anti-drug antibody levels of about 3.0 to about 100 ng/10 μL, about 3.0 to about 50 ng/10 μL, about 10 to about 100 ng/10 μL, about 10 to about 50 ng/10 μL, or about 20 to about 50 ng/10 μL. In some other embodiments, the phrase “high level of anti-drug antibody” includes anti-drug antibody levels of about greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ng/10 μl. 
     In certain aspects, the phrase “medium level of an anti-drug antibody” includes anti-drug antibody levels of about 0.5 to about 20 ng/10 μL, about 0.5 to about 10 ng/10 μl, about 2.0 to about 20 ng/10 μl, about 2.0 to about 10 ng/10 μL, about 2.0 to about 5.0 ng/10 or about 2.0 to about 5.0 ng/10 μl. 
     In certain aspects, the phrase “low level of an anti-drug antibody” includes anti-drug antibody levels of about 0.0 to about 5.0 ng/10 μl, about 0.1 to about 5.0 ng/10 μL, about 0.0 to about 2.0 ng/10 μL, about 0.1 to about 2.0 ng/10 μL, or about 0.5 to about 2.0 ng/10 μl. In other embodiments, the phrase “low level of anti-drug antibody” includes anti-drug antibody levels of about less than about 5.0, 4.0, 3.0, 2.0, 1.0, or 0.5 ng/10 μl. 
     II. Therapeutic Monitoring 
     Once the diagnosis or prognosis of a subject receiving anti-TNFα drug therapy has been determined or the likelihood of response to anti-TNFα drug has been predicted in an individual diagnosed with a disease and disorder in which TNFα has been implicated in the patho-physiology, e.g., but not limited to, shock, sepsis, infections, autoimmune diseases, RA, Crohn&#39;s disease, transplant rejection and graft-versus-host disease, according to the methods described herein, the present disclosure may further comprise recommending a course of therapy based upon the diagnosis, prognosis, or prediction. In certain instances, the present disclosure may further comprise administering to the individual a therapeutically effective amount of an anti-TNFα drug useful for treating one or more symptoms associated with disease and disorder in which TNFα has been implicated in the pathophysiology. For therapeutic applications, the anti-TNFα drug can be administered alone or co-administered in combination with one or more additional anti-TNFα drugs and/or one or more drugs that reduce the side-effects associated with the anti-TNFα drug (e.g., an immunosuppressive agent). Examples of anti-TNFα drugs are described herein include, but are not limited to, REMICADE™ (infliximab), INFLECTRA (Infliximab-dyyb), RENFLEXIS (Infliximab-abda), FLIXABI (Infliximab Biosimilar), REMSIMA (Infliximab Biosimilar), ENBREL™ (etanercept), HUMIRA™ (adalimumab), AMJEVITA (Adalimumab-atto), IMRALDI (Adalimumab Biosimilar), CYLTEZO (Adalimumab Biosimilar), HYRIMOZ (Adalimumab Biosimilar), HULIO (Adalimumab Biosimilar), CIMZIA® (certolizumab pegol), and other biologic agents. As such, the present disclosure advantageously enables a clinician to practice “personalized medicine” by guiding treatment decisions and informing therapy selection and optimization for anti-TNFα drugs such that the right drug is given to the right patient at the right time. 
     The anti-TNFα drug can be administered with a suitable pharmaceutical excipient as necessary and can be carried out via any of the accepted modes of administration. Thus, administration can be, for example, intravenous, topical, subcutaneous, transcutaneous, transdermal, intramuscular, oral, buccal, sublingual, gingival, palatal, intra joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, or by inhalation. By “co-administer” it is meant that an anti-TNFα drug is administered at the same time, just prior to, or just after the administration of a second drug (e.g., another anti-TNFα drug, a drug useful for reducing the side-effects of the anti-TNFα drug, etc.). 
     A therapeutically effective amount of an anti-TNFα drug may be administered repeatedly, e.g., at least 2, 3, 4, 5, 6, 7, 8, or more times, or the dose may be administered by continuous infusion. The dose may take the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, pills, pellets, capsules, powders, solutions, suspensions, emulsions, suppositories, retention enemas, creams, ointments, lotions, gels, aerosols, foams, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages. 
     An individual can also be monitored at periodic time intervals to assess the concentrations or levels of various antibodies. The antibody levels at various time points, as well as the rate of change of the antibody levels over time is significant. In certain instances, the rate of increase of one or more antibodies (e.g., autoantibodies to anti-TNFα antibodies) in an individual over a threshold amount indicates the individual has a significantly higher risk of developing complications or risk of side-effects. Information obtained from serial testing in the form of a marker velocity (i.e., the change in antibody levels over a time period) may be associated with the severity of the disease, the risk of complications of disease, and/or the risk of side-effects. 
     The methods of the present disclosure also provide for identifying primary non- or low-responders, e.g, for treatment with a therapeutic monoclonal antibody. These primary non- or low-responders may, for example, be patients that happen to have an innate or a pre-developed immunoglobulin response to the therapeutic agent. Where the therapeutic agent is a diagnostic antibody, the identification of primary non- or -low responders can ensure the selection of a suitable therapeutic agent for each individual patient. 
     III. Device 
     Various instruments and devices are suitable for use in the present disclosure. Many spectrophotometers have the capability to measure fluorescence. Fluorescence is the molecular absorption of light energy at one wavelength and its nearly instantaneous re-emission at another, longer wavelength. Some molecules fluoresce naturally, and others must be modified to fluoresce. 
     A fluorescence spectrophotometer or fluorometer, fluorospectrometer, or fluorescence spectrometer measures the fluorescent light emitted from a sample at different wavelengths, after illumination with light source such as a xenon flash lamp. Fluorometers can have different channels for measuring differently-colored fluorescent signals (that differ in their wavelengths), such as green and blue, or ultraviolet and blue, channels. In one aspect, a suitable device includes an ability to perform a time-resolved fluorescence resonance energy transfer (TR-FRET) experiment. 
     Suitable fluorometers can hold samples in different ways, including cuvettes, capillaries, Petri dishes, and microplates. The assays described herein can be performed on any of these types of instruments. In certain aspects, the device has an optional microplate reader, allowing emission scans in up to 384-well plates, Others models suitable for use hold the sample in place using surface tension. 
     Time-resolved fluorescence (TRF) measurement is similar to fluorescence intensity measurement. One difference, however, is the timing of the excitation/measurement process. When measuring fluorescence intensity, the excitation and emission processes are simultaneous: the light emitted by the sample is measured while excitation is taking place. Even though emission systems are very efficient at removing excitation light before it reaches the detector, the amount of excitation light compared to emission light is such that fluorescent intensity measurements exhibit elevated background signals. The present disclosure offers a solution to this issue. Time resolve FRET relies on the use of specific fluorescent molecules that have the property of emitting over long periods of time (measured in milliseconds) after excitation, when most standard fluorescent dyes (e.g. fluorescein) emit within a few nanoseconds of being excited. As a result, it is possible to excite cryptate lanthanides using a pulsed light source (e.g., Xenon flash lamp or pulsed laser), and measure after the excitation pulse. 
     As the donor and acceptor fluorescent compounds attached to antibody 1 and 2 move closer together, an energy transfer is caused from the donor compound to the acceptor compound, resulting in a decrease in the fluorescence signal emitted by the donor compound and an increase in the signal emitted by the acceptor compound, and vice-versa. The majority of biological phenomena involving interactions between different partners will therefore be able to be studied by measuring the change in FRET between 2 fluorescent compounds coupled with compounds which will be at a greater or lesser distance, depending on the biological phenomenon in question. 
     The FRET signal can be measured in different ways: measurement of the fluorescence emitted by the donor alone, by the acceptor alone or by the donor and the acceptor, or measurement of the variation in the polarization of the light emitted in the medium by the acceptor as a result of FRET. One can also include measurement of FRET by observing the variation in the lifetime of the donor, which is facilitated by using a donor with a long fluorescence lifetime, such as rare earth complexes (especially on simple equipment like plate readers). Furthermore, the FRET signal can be measured at a precise instant or at regular intervals, making it possible to study its change over time and thereby to investigate the kinetics of the biological process studied. 
     In certain aspects, the device disclosed in PCT/IB2019/051213, filed Feb. 14, 2019 is used, which is hereby incorporated by reference. That disclosure in that application generally relates to analyzers that can be used in point-of-care (POC) settings to measure the absorbance and fluorescence of a sample with minimal or no user handling or interaction. The disclosed analyzers provide advantageous features of more rapid and reliable analyses of samples having properties that can be detected with each of these two approaches. For example, it can be beneficial to quantify both the fluorescence and absorbance of a blood sample being subjected to a diagnostic assay. In some analytical workflows, the hematocrit of a blood sample can be quantified with an absorbance assay, while the signal intensities measured in a FRET assay can provide information regarding other components of the blood sample. 
     One apparatus disclosed in PCT/IB2019/051213 is useful for detecting an emission light from a sample, and absorbance of a transillumination light by the sample, which comprises a first light source configured to emit an excitation light having an excitation wavelength. The apparatus further comprises a second light source configured to transilluminate the sample with the transillumination light. The apparatus further comprises a first light detector configured to detect the excitation light, and a second light detector configured to detect the emission light and the transillumination light. The apparatus further comprises a dichroic mirror configured to (1) epi-illuminate the sample by reflecting at least a portion of the excitation light, (2) transmit at least a portion of the excitation light to the first light detector, (3) transmit at least a portion of the emission light to the second light detector, and (4) transmit at least a portion of the transillumination light to the second light detector. 
     One suitable cuvette for use in the present disclosure is disclosed in PCT/IB2019/051215, filed Feb. 14, 2019. One of the provided cuvettes comprises a hollow body enclosing an inner chamber having an open chamber top. The cuvette further comprises a lower lid having an inner wall, an outer wall, an open lid top, and an open lid bottom. At least a portion of the lower lid is configured to fit inside the inner chamber proximate to the open chamber top. The lower lid comprises one or more (e.g., two or more) containers connected to the inner wall, wherein each of the containers has an open container top. In certain aspects, the lower lid comprises two or more such containers. The lower lid further comprises a securing means connected to the hollow body. The cuvette further comprises an upper lid wherein at least a portion of the upper lid is configured to fit inside the lower lid proximate to the open lid top. 
     IV. Examples 
     Example 1 illustrates the measurement of detection of anti-TNFα drug biologics in blood and other matrices. 
     In this example, a solution phase homogenous time resolved FRET assay is used to detect drug levels of anti-TNF biologics in matrices such as blood or feces is described. Fluorescence resonance energy transfer (FRET) is a process in which a donor molecule in excited state transfers its excitation energy through dipole-dipole coupling to an acceptor fluorophore, when the two are brought into close proximity (typically less than 10 nm). 
     Upon excitation at a characteristic wavelength the energy absorbed by the donor is transferred to the acceptor, which in turn emits the energy. The level of light emitted from the acceptor fluorophore is proportional to the degree of donor acceptor complex formation. Biological sample materials are prone to autofluorescence, which can be minimized by utilizing time-resolved fluorometry (TRF). TRF takes advantage of unique rare earth elements called lanthanides, such as europium and terbium, which have exceptionally long fluorescence emission half-lives. Time-resolved FRET (TR-FRET) unites the properties of TRF and FRET, which is especially advantageous when analyzing biological samples. 
     If the TNF-alpha molecule is labeled with a donor fluorophore and an anti-TNF drug Fab fragment labeled with an acceptor fluorophore, TR-FRET can occur in the presence of anti-TNF-alpha drug ( FIG.  1 B ). In addition, each Fab region on the analyte (adalimumab) can contain the TNF-α donor and Anti-adalimumab Fab acceptor in a close proximity, enabling greater energy transfer producing an Amplified FRET signal. The increase in FRET signal of the acceptor is proportional to the amount of antibody drug present in the patients&#39; blood or feces as interpolated from a known amount of calibrators. 
       FIG.  2    illustrates a trFRET adalimumab (DRUG) calibration curve. Whole blood samples are serially diluted and used as calibrators. 
     A donor fluorophore H22TRENIAM-5LIO-NHS (structure in  FIG.  3   ) can be used to label TNF-alpha. The acceptor molecules that can be used include but are not limited to: AlexaFluor 488, AlexaFluor 546 and AlexaFluor 647.  FIG.  4    shows the structure of AlexaFluor 647. Lumi4 has 4 spectrally distinct peaks, at about 490 nm, about 545 nm about 580, and about 620 nm ( FIG.  5   ), which can be used for energy transfer. Donor and acceptor fluorophores are conjugated using primary amines on TNF-alpha and the Fab fragment of an anti-TNF-alpha drug antibody respectively. 
       FIG.  6    illustrates a trFRET adalimumab (DRUG) assay reaches equilibrium after 3 minutes. Eight concentrations (0 μg/mL to 50 μg/ml) are tested. Reagents were mixed with a sample and read at different time points as shown. 
     Example 2 illustrates the measurement of detection of anti-TNFα drug autoantibodies. 
     Monoclonal antibodies against TNFα such as infliximab (IFX), adalimumab (HUMIRA™), and certolizumab have been shown to be effective in treating inflammatory bowel disease (IBD) and other inflammatory disorders. Anti-drug antibodies (autoantibodies) may reduce the drug&#39;s efficacy and/or induce adverse effects. However, autoantibodies have been found not only in patients treated with the chimeric antibody infliximab, but also in patients treated with the humanized antibody adalimumab. Monitoring of autoantibodies and drug levels in individual patients may help optimize treatment and dosing of the patient. 
       FIG.  7 A  illustrates a method of the disclosure for detecting anti-drug antibody (autoantibodies). An anti-TNFα drug such as infliximab (IFX) can be labeled with a donor and another infliximab (IFX) can be labeled with an acceptor. Once bound to the autoantibodies human anti-chimeric antibodies (HACA) and the donor is excited, FRET occurs. 
     Similarly, as shown in  FIG.  7 B  the present disclosure provides methods for detecting anti-drug antibody (autoantibodies) using the Fab portion of anti-TNFα drugs (e.g. such as infliximab (IFX)). A Fab portion of an anti-TNFα drug such as infliximab (IFX) can be labeled with a donor and another Fab portion of infliximab (IFX) can be labeled with an acceptor. Once bound to the autoantibodies human anti-chimeric antibodies (HACA) and the donor is excited, FRET occurs. 
     Example 3 illustrates the measurement of infliximab using the methods of this disclosure. 
     In this example, a standard curve for infliximab was generated using the methods described herein. A TNF-alpha molecule was labeled with a donor fluorophore and an anti-TNF drug Fab fragment was labeled with an acceptor fluorophore. TR-FRET occurred in the presence of infliximab. The increase in FRET signal of the acceptor is proportional to the amount of drug present in the patients&#39; sample. The results are tabulated below and shown in  FIG.  8   . 
     
       
         
           
               
               
               
               
               
               
               
               
             
               
                   
               
               
                   
                 Expected 
                   
                   
                   
                 Calculated 
                   
                   
               
               
                   
                 Conc. 
                 FRET 
                 Donor 
                 Acceptor 
                 Conc. 
                   
                 % 
               
               
                 Standards 
                 (ng/mL) 
                 Ratio 
                 RFU 
                 RFU 
                 (ng/mL) 
                 CV 
                 Recovery 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 CAL-1 
                 53959.7 
                 12620 
                 438013 
                 552727 
                 53983.5 
                 2.5% 
                 100.0% 
               
               
                 CAL-2 
                 20898.1 
                 6187 
                 456810 
                 282627 
                 20924.1 
                 1.0% 
                 100.1% 
               
               
                 CAL-3 
                 9026.1 
                 2971 
                 461801 
                 137188 
                 8938.5 
                 1.1% 
                 99.0% 
               
               
                 CAL-4 
                 4200.6 
                 1713 
                 471821 
                 80808 
                 4220.7 
                 1.2% 
                 100.5% 
               
               
                 CAL-5 
                 1839.5 
                 1124 
                 468687 
                 52671 
                 1971.2 
                 1.5% 
                 107.2% 
               
               
                 CAL-6 
                 836.6 
                 828 
                 463223 
                 38377 
                 819.7 
                 3.6% 
                 98.0% 
               
               
                 CAL-7 
                 2.9 
                 608 
                 466812 
                 28363 
                 0.0 
                 — 
                 — 
               
               
                   
               
            
           
         
       
     
     Example 4 illustrates a comparison of measuring infliximab using the methods of this disclosure versus Homogenous Mobility Shift Assay (HMSA). 
     The samples were measured using the inventive method and compared to the measurements using HMSA. The measurements using the inventive methods were performed in duplicate. The results are shown in  FIG.  9   . A R2 of 1 indicates that the regression predictions perfectly fit the data. Here, the R2 is equal to 0.9759 showing excellent correlation of the inventive methods with the comparator. 
     Example 5 illustrates the measurement of adalimumab using the methods of this disclosure. 
     In this example, a standard curve for adalimumab was generated using the methods described herein. A TNF-alpha molecule was labeled with a donor fluorophore and an anti-TNF drug Fab fragment was labeled with an acceptor fluorophore. TR-FRET occurred in the presence of infliximab. The increase in FRET signal of the acceptor is proportional to the amount of drug present in the patients&#39; sample. The results are tabulated below and shown in  FIG.  10   . 
     
       
         
           
               
               
               
               
               
               
               
               
             
               
                   
               
               
                   
                 Expected 
                   
                   
                   
                 Calculated 
                   
                   
               
               
                   
                 Conc. 
                 FRET 
                 Donor 
                 Acceptor 
                 Conc. 
                   
                 % 
               
               
                 Standards 
                 (ng/mL) 
                 Ratio 
                 RFU 
                 RFU 
                 (ng/mL) 
                 CV 
                 Recovery 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 CAL-1 
                 49088.9 
                 18904 
                 432999 
                 818564 
                 49133 
                 2.6% 
                 100.1% 
               
               
                 CAL-2 
                 19891.8 
                 8889 
                 442802 
                 393588 
                 19921 
                 0.5% 
                 100.1% 
               
               
                 CAL-3 
                 9985.7 
                 4271 
                 458301 
                 195665 
                 9831 
                 2.5% 
                 98.4% 
               
               
                 CAL-4 
                 4575.1 
                 2199 
                 458552 
                 100816 
                 4862 
                 1.1% 
                 106.3% 
               
               
                 CAL-5 
                 2513.4 
                 1282 
                 451824 
                 57918 
                 2432 
                 1.3% 
                 96.8% 
               
               
                 CAL-6 
                 1335.5 
                 914 
                 460149 
                 42036 
                 1237 
                 0.0% 
                 92.6% 
               
               
                 CAL-7 
                 0.0 
                 525 
                 457238 
                 23996 
                 39 
                 — 
                 — 
               
               
                   
               
            
           
         
       
     
     Example 5 illustrates a comparison of measuring adalimumab (ADA) using the methods of this disclosure versus Homogenous Mobility Shift Assay (HMSA). 
     The samples were measured using the inventive method and compared to the measurements using HMSA. The measurements using the inventive methods were performed in duplicate. The results are shown in  FIG.  11   . A R2 of 1 indicates that the regression predictions perfectly fit the data. Here, the R2 is equal to 0.9535 showing excellent correlation of the inventive methods with the comparator. 
     Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.