Sensitivity improvement of an immunoassay via branched DNA

Abstract

Methods of detecting analytes in a sample are described herein using chemiluminescent labels and branched DNA. Solid supports, reagents, and compounds for use in these methods are also described. Typically, the methods involve specific assay formats which provide the requisite high resolution for detecting low concentrations of analytes such as exosome protein biomarkers in samples.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to and the benefit of U.S. App. No. 63 / 649,049, filed May 17, 2024, the entirety7 of which is hereby incorporated by reference in its entirety7. SEQUENCE LISTING

[0002] The present application contains a sequence listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The Sequence Listing XML file is named 2024P09519WO-SL.xml, was created on May 15, 2025, and is 34,495 bytes in size. FIELD OF DISCLOSURE

[0003] The present disclosure relates to high resolution assay methods for detection or measurement of analytes ty pically present in low concentrations in a biological sample. These assay methods typically involve the use of branched DNA with a an acridiniums conjugated to a label probe. BACKGROUND

[0004] Detection of analytes of interest in certain biological samples is often hampered by detection limits of immunoassays. Certain neurological diseases, disorders, and conditions are associated with biomarkers that could allow for the improvement of diagnostic accuracy and prognostic assessment.

[0005] Exosome protein biomarkers are proteins that are carried by exosomes, which are small vesicles secreted by various cells. Exosomes can transfer information and molecules between cells and reflect the state and function of the original cells. Therefore, exosome protein biomarkers have potential clinical value for diagnosis, prognosis, and treatment of various diseases.

[0006] According to some recent studies, exosome protein biomarkers can be used to detect and monitor lung cancer, Alzheimer’s disease, and other cancers. For example, Glypican-1 (GPC1), a cell surface proteoglycan, is enriched in exosomes derived from pancreatic cancer cells and can be detected in the serum of patients3. GPC1-expressing exosomes can distinguish pancreatic cancer patients from healthy individuals and chronic pancreatitis patients with high accuracy. Another example is neuron-derived exosomes (NDEs) from plasma, which contain phosphorylated tau (p-tau), amyloid beta (AP), and neurofilament light chain (NFL) proteins that are associated with Alzheimer’s disease.

[0007] Neurological protein biomarkers are proteins can help diagnose, monitor, or predict neurological disorders or be used to evaluate the effects of therapeutic interventions on the disease process. Neurological protein biomarkers have a high clinical value, as they can provide objective and reliable information about the brain function and pathology7, which are often difficult to assess by conventional methods. For example, cerebrospinal fluid (CSF) biomarkers for Alzheimer’s disease may include amyloid-beta (AP). total tau. and phosphorylated tau proteins, which reflect the amyloid and tau pathology in the brain. CSF biomarkers can help diagnose Alzheimer’s disease at an early stage, differentiate it from other ty pes of dementia, and monitor the response to anti-amyloid or anti-tau therapies. However, they are often present in low concentrations in biological samples making assaying for their presence (or concentration) in a sample difficult.

[0008] NDEs from plasma can discriminate Alzheimer’s disease patients from healthy controls and mild cognitive impairment patients with high sensitivity and specificity7. Although exosome protein biomarkers have several advantages over conventional biomarkers, such as being more stable, accessible, and representative of the disease state. However, there are also some challenges and limitations in their clinical applications, especially, the amount of exosomal proteins is low, and often can hardly be detected with a conventional clinical diagnostic immunoassay platform. In addition, neurological protein biomarkers also can hardly be detected on conventional clinical diagnostic immunoassay platform.

[0009] Typically, the clinical laboratory defines lower limits of most assays as the lower limit of quantitation (LLOQ) or limit of detection (LOD). Lower limit of quantitation refers to the lowest value at which an accurate quantitative value (CV < 20%) can be reported, while the LOD is the value at which the assay is able to distinguish between the presence or absence of an analyte. Certain analytes having low concentration such as NfL cannot be measured with most immunoassays due to lower limits of detection intrinsic in typical immunoassay formats.

[0010] Certain assay formats have been developed in an attempt to afford better detection capability of anayltes of interest in a sample. For example, Proximity Extension Assays (PEA) analyze proteins through target amplification. Each of the oligonucleotide antibody-pairs contains unique DNA sequences allowing hybridization only to each other. Subsequent proximity extension will create the unique DNA reporter sequences which are amplified by real-time PCR. However, cross-reactive events will not be detected with the proximity extension assay panels since only matched DNA reporter pairs can hybridize to produce an amplicon for next generation sequencing (NGS) or real-time quantitative polymerase chain reaction (qPCR).

[0011] Another assay format designed to increase the sensitivity of an immunoassay is the Single Molecule Array (SiMoA). In these formats, paramagnetic particles are coupled with an antibody designed to bind a specific protein target and form a traditional immunocomplex. The beads are subsequently concentrated and washed. When low abundance biomarkers are present, there may be more beads than targeted proteins. Each bead may contain one or less bound protein. The beads are then loaded into microarrays in the presence of substrate. The array consists of microwells dimensioned to hold a single bead (the wells may be femtolitersized). An oil solution is added to seal each well and remove excess beads. Within each well, a single target molecule generates enough fluorescence signal to be measured and positive wells are enumerated to determine protein concentration.

[0012] However, both technologies can hardly be integrated into current clinical diagnostic platforms installed worldwide due to its batch process.

[0013] There is a continuing need for assays that can provide high sensitivity measurements of analyte detection-particularly for analytes that are typically present in low concentrations of biological samples. SUMMARY

[0014] In accordance with the foregoing objectives and others, the present disclosure includes methods for the detection of analytes in a sample through the use of acridinium conjugates to analytes or binding partners for analytes such as antibodies using branched DNA platforms which may afford detection of analytes in low concentrations.

[0015] The general concept of the branched DNA assays of the present disclosure typically begins with the fixation and permeabilization of an analyte to a preamplifier oligomer such as an oligonucleotide. The preamplifier may behave as the trunk of the branched amplification network to form. Amplifier strands are then added, hybridizing to the pre-amplifier strand and providing branches for the binding of many labeled probe oligos. Multiple probe oligos can bind to each preamplifier, creating one large network that may amplify the signal for detecting the underlying target analyte. In some embodiments, the probe oligo are conjugated to a chemiluminescent acridinium.

[0016] Methods for the detection or quantification of an analyte in a sample (e.g., a biological sample such as blood, saliva, serum, a sample derived from a biological sample such as a diluted biological sample) are provided which may comprise one or more of: (a) mixing the sample with a composition comprising a branched DNA (b-DNA) pre-amplifier oligomer conjugated to a first antibody or antibody fragment that binds to the analyte and optionally a particle having a second antibody or antibody fragment that binds to the analyte attached to the surface of the particle; (b) adding to the mixture a solid support (e.g., particle, chip) having a second antibody or antibody fragment that binds to the analyte attached to the surface of the particle (e.g., if the particle is not already present in the mixture); (c) adding to the mixture a b-DNA amplifier oligomer that binds to the preamplifier oligomer, wherein, optionally, multiple amplifier oligomers bind to at least one of the pre-amplifier oligomers conjugated to the analyte; (d) adding to the mixture a label probe comprising a chemiluminescent moiety bound to a probe oligomer, wherein the probe oligomer binds to one of the amplifier oligomers, wherein, optionally, multiple probe oligomers bind to a single amplifier oliogomer; (e) preparing the mixture to measure chemiluminescence (e.g., by separation of the solid support (e.g.. particle) having chemiluminescent acridiniums conjugated to the surface from the mixture); triggering chemiluminescence from the preparation (e.g., a washed branched DNA probe complex conjugated to the solid support (e.g., particle); and (g) detecting the presence or calculating the concentration of said at least one analyte by comparing the amount of chemiluminescence with a standard dose response curve which relates the amount of light emitted to a known concentration of the at least one of the multiple analytes.

[0017] In some embodiments, the method may comrpise one or more of: (a) mixing the sample with a solid support (e.g., particle, chip) having a second antibody or antibody fragment that binds to the analyte attached to the surface of the particle (e.g.. if the particle is not already present in the mixture); (b) adding to the mixture a composition comprising a branched DNA (b-DNA) preamplifier oligomer conjugated to a first antibody or antibody fragment that binds to the analyte; (c) adding to the mixture a b-DNA amplifier oligomer that binds to the preamplifier oligomer, wherein, optionally, multiple amplifier oligomers bind to at least one of the pre-amplifier oligomers conjugated to the analyte; (d) adding to the mixture a label probe comprising a chemiluminescent moiety bound to a probe oligomer, wherein the probe oligomer binds to one of the amplifier oligomers, wherein, optionally, multiple probe oligomers bind to the amplifier oligomer; (e) preparing the mixture to measure chemiluminescence (e.g., by separation of the particle having chemiluminescent acridiniums conjugated to the surface from the mixture); (f) triggering chemiluminescence from the preparation (e.g., a washed branched DNA probe complex conjugated to the solid support (e.g., particle); and (g) detecting the presence or calculating the concentration of said at least one analyte by comparing the amount of chemiluminescence with a standard dose response curve which relates the amount of light emitted to a known concentration of the at least one of the multiple analytes.

[0018] Following formation of the branched DNA probe complex conjugated to the particle via the analyte, the sample is typically prepared to induce chemiluminesence in a manner that the analytes can be measured and / or their concentration quantified. For example, the preparing step may comprise: (el) providing a branched DNA probe complex conjugated to the particle via the analyte in a mixture, (e2) separating said branched DNA probe complex conjugated to the particle support from said mixture; and (e3) optionally washing the branched DNA probe complex conjugated to the particle.

[0019] In some embodiments, the preparing step may comprise separating the particle (complexed with the label) from the mixture and the chemiluminescence is triggered from the particle or the separated mixture. In some embodiments, the method further comprises incubating the mixture prior to adding the particle. In some embodiments, the incubating prior to addition of the solid phase comprises heating the mixture for more than 30 minutes (e.g., from 30 minutes to 120 minutes, from 30 minutes to 60 minutes). In some embodiments, the method further comprises incubating the mixture after adding the particle. In some embodiments, this incubation after particle addition may involve heating the mixture for less than 30 minutes (e.g., from 10 minutes to 30 minutes, from 10 minutes to 20 minutes).

[0020] In some embodiments, the the analyte is an exosome (e.g., neuron-derived, astrocyte-derived, oligodendrocyte-derived, microglia-derived) protein biomarker. For example, the analyte may be glypican-1 (GPC1), neurofilament light chain (NFL), phosphorylated tau (p-tau), or amyloid beta (A|3). The sample may be blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cervical swab, tears, saliva, buccal swab, skin, brain tissue, or cerebrospinal fluid, or derived therefrom (e.g., diluted sample). In some embodiments, the preparing step comprises separating the particle from the mixture and the chemiluminescence is triggered from the particle or the separated mixture. In some embodiments, the method further comprising a washing step between the mixing step and any of the adding steps (e.g., after mixing the sample the sample with particle). In some embodiments, the label probe is added in molar excess of pre-amplifier oligomer (e.g., less than 100x excess or less than 50* excess or less than 40x excess or less than 30x excess or from 5x excess to 25x excess).

[0021] Immunoassay compositions are also provided which may be used in the methods of the present disclosure. For example, the composition may comprise a chemiluminescent label conjugated to a probe oligomer; and a carrier or excipient. In various implementations, the composition further comprises a buffer.

[0022] Products formed from the methods of the present disclosure are provded. For example, a solid particle is disclosed, wherein the solid particle is coated with streptavidin conjugated to a biotinylated antibody for an analyte optionally through a linker (e.g., PEG, lodo-PEG), wherein the antibody is bound to the analyte, and the bound analyte is further bound to a second antibody (e.g., monoclonal mouse antibody) or fragment thereof of the analyte, wherein the second antibody is conjugated to a pre-amplifier oligomer, the pre-amplifier oligomer is hybridized to one or more amplifier oligomers; and the amplifier oligomer is hybridized to one or more probe labels comprising a chemiluminescent moiety (e.g., chemiluminescent acridinium) conjugated to a probe oligomer. Conjugation of the chemiluminescent moiety to the oligomer (e. probe label) may be at a terminal sugar in the sequence (e.g., 3', 5'). Conjugation may occur at, for example, a hydroxyl group in the deoxyribose sugar and phosphate group backbone.

[0023] In various implementations, the first antibody or antibody fragment is a mouse monoclonal antibody or fragment thereof (e.g., F(ab)).

[0024] The general concept of the branched DNA assays of the present disclosure typically begins with the fixation and permeabilization of an analyte to a preamplifier oligomer such as an oligonucleotide or oligopeptide. The preamplifier may behave as the trunk of the branched amplification network to form. Amplifier strands are then added, hybridizing to the preamplifier strand and providing branches for the binding of many labeled probe oligos. Multiple probe oligos can bind to each preamplifier, creating one large network that may amplify the signal for detecting the underlying target analyte. In some embodiments, the probe oligo is conjugated to a chemiluminescent acridinium.

[0025] The pre-amplifier, typically a polynucleotide, typically serves as an intermediate binding component between the solid phase and one or more amplifiers. Typically, the preamplifier hybridizes simultaneously to one or more target probe oligos ( and to a plurality of amplifiers. Exemplary pre-amplifiers are described, for example, in U.S. Pat. Nos. 5,635,352, 5,681,697 and 7,709,198 and U.S. publications 2008 / 0038725, 2009 / 0081688 and 2017 / 0101672, each of which is incorporated herein by reference in its entirety.

[0026] The amplifier which is typically a polynucleotide capable of hybridizing to multiple label probes and, optionally, one or more complementary regions on the preamplifier. Typically, the amplifier hybridizes to multiple identical label probes oligomers (which, are typically conjugated to one or more chemiluminescent acridiniums). The amplifier can also hybridize to a target nucleic acid, to at least one target probe of a pair of target probes, to both target probes of a pair of target probes, or to nucleic acid bound to a target probe such as an amplifier, pre-amplifier or pre-pre-amplifier. For example, the amplifier can hybridize to at least one target probe and to a plurality of label probes, or to a pre-amplifier and a plurality of label probes. The amplifier can be. for example, a linear, forked, comb-like, or branched nucleic acid. As described herein for all polynucleotides described herein, the amplifier can include modified nucleotides and / or nonstandard inter-nucleotide linkages as well as standard deoxyribonucleotides, ribonucleotides, and / or phosphodiester bonds. Suitable amplifiers are described, for example, in U.S. Pat. Nos. 5,635,352, 5,124,246, 5,710,264, 5,849,481, and 7,709,198 and U.S. publications 2008 / 0038725 and 2009 / 0081688, each of which is incorporated herein by reference in their entirety.

[0027] The branched DNA complexes are generally formed through the use of complementary sequences on the probe oligomer and the amplifier oligomer, as well as complementary sequences on the amplifier oligomer and the pre-amplifier oligomer. The length of the complementary seqeuences between any two pairs may be, for example, between 5 and 40 bases in length (e.g., between 10 and 25, between 15 and 25). Typically, the sequences are sequences of base pairs selected from adenine (A), thymine (T), guanine (G), cytosine (C). uracil (U, typically used if branched RNA technology is implemented), isocytosine (F), and isoguanine (J). For example, the preamplifier oligomer may have the structure: 5‘-TS-OL-(OL-CS '-OL)m-OL-TS-3 ’ the amplifier oligomer may have the structure: 5’-TS-OL-CSp-OL-(OL-CS2)n-OL-TS-3’ the probe oligomer may have the structure: 5’-TS-CS2*-TS-3’ where m and n are independently from 1-20 (e.g.. 2-10. 2-5) TS is independently at each occurence absent or a sequence of nucleic acid residues (e.g., of from 1-15 bases in length) and may cap the indicated oligomer (e.g., preamplifier oligomer, amplifier oligomer, probe oligomer); OL is independently at each occurrence absent or a sequence of nucleic acid residues (e.g., of from 1-10 bases in length); CS1 is a complementary sequence of CS1*; and CS2 is a complementary sequence of CS2*. In certain aspects, at least one of TS or OL is, independently, (T)i-io. Typically, the oligomers (e.g., probe oligomer, pre-amplifier oligomer, amplifier oligomer) of the present disclosure have a melting temperature of between 30°C and 70°C (e.g., between 40°C and 60°C). In various implementations, any oligomer does not form a hairpin structure. In certain aspects, any oligomer used does not have any selfcomplementarity. In various aspects, the degree of intra or inter self-complementarity is less than 50% across the full length of the oligonucleotide. For example, CS1 or CS1* may be AGT FAJ CGC FGT AF (SEQ ID NO 1) and the other of CS1 or CS1* may be TCA JTF GCG JCA TJ (SEQ ID NO 2). In some embodiments, CS2 or CS2* may be AGT FAJ CGC FGT AF (SEQ ID NO 1) and the other of CS2 or CS2* may be TCA JTF GCG JCA TJ (SEQ ID NO 2). CS1 or CS1* may be CFT AGJ GFC GTJ GA (SEQ ID NO 3) and the other of CS1 or CS1* may be GJA TCF CJG CAF CT (SEQ ID NO 4). In some embodiments, CS2 may be CFT AGJ GFC GTJ GA (SEQ ID NO 3) and CS2* may be GJA TCF CJG CAF CT (SEQ ID NO 4). CS1 or CS1* may be AGG CAT AGG ACC CGT GTC (SEQ ID NO 5) and the other of CS1 or CS1* may be TCC GTA TCC TGG GCA CAG (SEQ ID NO 6). In some embodiments, CS2 or CS2* may be AGG CAT AGG ACC CGT GTC (SEQ ID NO 5) and the other of CS2 or CS2* may be TCC GTA TCC TGG GCA CAG (SEQ ID 6). CS1 or CS1* may be GCA CTT GGT ACG GCG CTG ACT (SEQ ID NO 7) and the other of CS1 or CS1* may be CGT GAA CCA TGC CGC GAC TGA (SEQ ID NO 8). CS2 or CS2* may be GCA CTT GGT ACG GCG CTG ACT (SEQ ID NO 7) and the other of CS2 or CS2* may be CGT GAA CCA TGC CGC GAC TGA (SEQ ID NO 8).

[0028] In some embodiments, the probe oligomer is a an oligonucleotide comprising (or being) the sequence 5-AGT FAJ CGC FGT AFT T-3‘ (SEQ ID NO 9) or a sequence having more than 80% (e.g., more than 90%, more than 95%) identity therewith; wherein F is isocytosine and J is isoguanine. For example, the probe oligomer may be: 5-AGT FAJ CGC FGT AFT T-3‘- (SEQ ID NO 9) linked (e.g., via a linker) to a chemiluminescent moiety (e.g., at the 3’ end). In various implementations, the amplifier oligomer is an oligonucleotide comprising (or being) the sequence 5'-TTT-(JTA CJG CGF TJA CT TTTTT)n-TCF ACG JCF CTA JG-3' (SEQ ID NO 10) or a sequence having more than 80% (e.g., more than 90%, more than 95%) identity' therewith; wherein n is an integer (e.g., an integer from 1-50 or 1-20 or 2-50 or 2-20 or 2-15 or 5-15); F is isocytosine and J is isoguanine. In certain embodiments, the pre-amplifier oligomer is an oligonucleotide having (or being) the sequence 5 '-TTT-(CFT AGJ GFC GTJ GA TTTTT)n-3' (SEQ ID NO 11) or a sequence having more than 80% (e.g., more than 90%, more than 95%) identity therewith; wherein n is an integer (e.g.. an integer from 1-50 or 1-20 or 2-50 or 2-20 or 2-15 or 5-15): F is isocytosine and J is isoguanine.

[0029] In some embodiments, the probe oligomer is a an oligonucleotide comprising (or being) the sequence 5-GCA CTT GGT ACG GCG CTG ACT TTT-AE-3’ (SEQ ID NO 12) or a sequence having more than 80% (e.g., more than 90%, more than 95%) identity therewith. For example, the probe oligomer may be: 5-GCA CTT GGT ACG GCG CTG ACT TTT- 3‘ (SEQ ID NO 12) linked (e.g., via a linker) to a chemiluminescent moiety (e.g., at the 3’ end). In various implementations, the amplifier oligomer is an oligonucleotide comprising (or being) the sequence 5‘ -TGA CAC GGG TCC TAT GCC TTT TAG TCA GCG CCG TAC CAA GTG CTT TTT TAG TCA GCG CCG TAC CAA GTG CTT TTT TAG TCA GCG CCG TAC CAA GTG CTT TTT T-3‘ (SEQ ID NO 13) or a sequence having more than 80% (e.g., more than 90%, more than 95%) identity therewith. In certain embodiments, the pre-amplifier oligomer is an oligonucleotide having (or being) the sequence 5 - AGG CAT AGG ACC CGT GTC TTT TTT AGG CAT AGG ACC CGT GTC CGT GGA TGT TTG AGG CAT AGG ACC CGT GTC TTT TTT -3' (SEQ ID NO 14) or a sequence having more than 80% (e.g.. more than 90%, more than 95%) identity therewith. For example, the pre-amplifier oligomer may have the sequence: 5‘ -TTT GGA AAG AAA GTG AAG TGT AGG CAT AGG ACC CGT GTC TTT TTT AGG CAT AGG ACC CGT GTC CGT GGA TGT TTG AGG CAT AGG ACC CGT GTC TTT TTT- 3‘ (SEQ ID NO 15).

[0030] In various implementations, the chemiluminescent moiety is an acridium or an alkaline phosphatase. The label probe may be formed by i) reacting a chemiluminescent moiety (e.g., acridinium, alkaline phosphatase) compound comprising a reactive functional group with a linking compound (e.g., a compound comprising a linker with reactive functional groups on each end, a compound already conjugated to the probe oligomer) and ii) reacting the probe oligomer to the linking compound. The chemiluminescent acridinium compound comprising a reactive functional group may have the structure: RFGLT                            (I) wherein RFG is a reactive functional group for conjugation to the probe oligomer, L is absent (i.e., it is a bond) or a linker optionally comprising a group Lc or ZL, and ¥ is a chemiluminescent acridinium comprising the structure: and are independently 0 (e.g., all R2 groups are hydrogen, all Rs groups are hydrogen), 1,2, 3, or 4; Ri is hydrogen, -R, -Xb, -RL-Xb, -Lc-R, -Lc-Xb (e.g., -Li-Xb), -Z, -RL-Z, -Lc-Z (e.g., -Li-Z), or -Rl-Lc-Rl-Z (e.g., -Rl-Li-Rl-Z); R2 and Rs are independently selected at each occurrence from hydrogen, R, an electron donating group, -Xc. -RL-XC, -Lc-Xc (e.g.. -Li-Xc), and -Z; wherein two vicinal R2 or R3 groups may together form a fused cyclic group (e.g., 5-7 membered fused aryl or heteroaryl group, 5-7 membered fused heterocyclic group) and wherein R2 or R3 may comprise a linkage to an imaging agent such as a fluorophore (e.g., rhodamine); Lc is a divalent C1-35 alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted (e.g., with 1 to 20 heteroatoms, with 1-20 substituents); ZL is a zwitterionic linker group having the structure: Xa ( rL^ \ A R> . “m” is 0 (i.e. it is a bond) or 1; “n” and “ / 2” are independently at each occurrence an integer from 0 (i.e. it is a bond) to 10; Z is a zwitterionic group independently at each occurrence has the structure: “<7” and ‘7” are independently 0 or 1; “r” is independently an integer from 0 to 10 (e.g., from 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); Xa and Xb are independently at each occurrence an anionic group; Xc is a protonated anionic group; Li is independently at each occurrence -0-, -S-, -NH-, -N(Rn)-, -(CH2)i-io-, -S(=O)i-2--C=C- -C=C-(CH2) 1-3-, -C(O)-, -O-C(O)-, -C(O)-(CH2) 1-4-, -(CH2) i-4-C(O)-, C(O) O , C(O) N(Rn) , C(O) NH , N(Rn) C(O) , NH C(O) , -C(O)-N(Rn)-(CH2)i-3-,       -(CH2)i-3-C(O)-N(Rn)-       -(CH2)i-3-N(Rn)-C(O)-, -NH-S(O)i-2-. -N(Rn)-S(O)i-2-, -S(O)i-2-N(Rn)- -S(O)i-2-NH-. -(CH2)i-3-NH-S(O)i-2--(CH2)i-3-N(Rn)-S(O)i-2-,      -(CH2)i-3-S(O)i-2-N(Rn)-      -(CH2)i-3-S(O)i-2-NH- -O-(CH2)i-4-,    -(CH2)i-4-O-,    -S-(CH2)i-4-    -(CH2)i-4-S-,    -NH-(CH2)i-4- -N(Rn)-(CH2)i-4-, -(CH2)i-4-N(Rn)- -(OCH2)i-io- -(CH20)i-io-, -(OCH2CH2)i-io- or -(CH2CH20)i-io-; Rl is independently at each occurrence a C1-20 bivalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, phenyl, mono alkyl substituted phenyl, di alkyl substituted phenyl, alkynyl. arylalkyl), optionally having one or more (e.g, 1-10, 1-5) points of substitution (e.g, with 1-10 heteroatoms, with 1-10 substituents); R is independently at each occurrence hydrogen or C1-35 hydrocarbon (e.g., alkyl, alkenyl, alky nyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms, with 1-20 substituents); R’ and R” are independently at each occurrence hydrogen or a Ci-io alkyl; Rn is independently at each occurrence from hydrogen or C1-5 alkyl (e.g, methyl, ethyl, propyl); and R’ is hydrogen or a Ci-io alkyl; or a salt thereof (e.g., a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxylate salt).

[0031] The chemiluminescent acridinium comprising a reactive functional group may have the structure of formula (la): (la) wherein Q is O or N: Y is selected from -R or -RL-Z, or in the case where Q is 0 then Y is absent; and Y’ is either absent (i.e. it is a bond), or is selected from -Li-, -RL-, -Rl-Li-, -Li-Li-, -Li-R1 -. -Li-R1 -Li, and -R1 -Li-R1 - The chemiluminescent acridinium comprising a reactive functional group may have the structure of formula (lb) or (Ic): (lb) (Ic) wherein R4-R7 are independently hydrogen, an electron donating group, or C1-35 alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, or amino; and Y” is either absent (i.e., it is abend) or-Lc-, -Li- -RL-, or-RL-Li-.

[0032] In some embodiments, L is a C1-C5 alkylene. In various implementations, the reactive functional group of said chemiluminescent acridinium comprising a reactive functional group is an N-succinimidyl ester. In certain embodiments, R2 and R3 are independently -Xc, -RL-Xc, -Lc-Xc (e.g., -Li-Xc). In some embodiments, R2 and R3 are independently alkoxy (e.g., C1-C4 alkoxy) substituted with -C(O)OH. -SO2OH), -OSO2OH), -OP(O)(ORp)OH, -OH, or combinations thereof.

[0033] For example, the probe oligomer and chemiluminescent moiety may have the structure: wherein RFG* is the linker formed by reacting the reactive functional group with the terminal hydroxyl group on the oligonucleotide at either the 3’ or 5’ end. For example, RFG* may be -OP(O)(OR)O-, where R is hydrogen or alkyl.

[0034] The method may be used to characterize a disease, disorder, or condition. For example, if the analyte is a neuronal biomarker such as neurofilament light chain (NFL), phosphorylated tau (p-tau), or amyloid beta (AP) a subject from which the sample is taken may be diagnosed with a neuronal disease (e.g., amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer’s disease, and Huntington’s disease) if the analyte level is above a certain concentration in the blood (e.g., between 1 pg / mL-20 pg / mL). In some embodiments, the method provides the requisite resolution to track progression of the disease, disorder, or condition. For example, the concentration of the analyte may be measured from a first sample taken from a subject. After a time period, a second biological sample may be taken from the subject and the concentration may be determined. Changes in the biomarker level may be correlated with disease progression.

[0035] In certain implementations, the chemiluminescent label is conjugated to a first immunoglobulin antibody fragment (e.g., F(ab), F(c)). In particular embodiments, the chemiluminescent label is conjugated to F(ab) fragments which may still bind to antigens or analytes but is monovalent (and without the F(c) portion). In some embodiments, the first antibody or antibody fragment is a mouse monoclonal antibody or fragment thereof (e.g, F(ab)). In some embodiments, the linker between the antibody or fragment thereof and the carrier protein comprises (or is) polyethylene glycol (PEG) which may be independently covalent linked to both moieties. For example, the polyethelene glycol linker may have from 2-20 (e.g., 2-10, 2-5) ethylene glycol units.

[0036] The assays of the present disclosure are typically a 2-step sandwich immunoassay using acridinium ester chemiluminescent and branched DNA technology. The first antibody, in the Lite Reagent, may be a mouse monoclonal antibody (or a fragment thereof) conjugated to the pre-amplifier oligomer. The second antibody may be a biotinylated mouse monoclonal antibody (or a fragment thereof) bound to streptavidin-coated paramagnetic microparticles in the Solid Phase.

[0037] By using the conjugates and assay formats described herein, high resolution assays may be performed. These assays may be performed on analytes typically having previously thought undetectable concentrations in biological samples such as those analytes that cross the blood brain barrier. Specific incubation and sequential steps as described herein may result in these assay formats with increased resolution. For example, the detecting step may detect a difference in concentration of less than (or from 0.005 pg / mL to) 5 pg / mL (e.g., less than 1 pg / mL, less than 0.5 pg / mL, less than 0.2 pg / mL, from 0.01-0.2 pg / mL). In various implementations, the assays of the present disclosure may be characterized as having higher signal (e.g., as measured by chemiluminescent RLU) without increased noise. For example, the presently disclosed assays may have a noise level (e.g., as measured by chemiluminescent RLU) comparable (e.g., within 10% or within 5% or within 1%) of an otherwise identical sandwich immunoassay having conjugation of a single chemiluminescent moiety to the target in the solid support complex. In some embodiments, the assays of the present disclosure have a signal to noise (S / N) of more than (e.g., up to 500) 10 or more than 50 or more than 75 or more than 100 or more than 125.

[0038] Kits for the detection of an analyte are also provided. The kit may comprise an immunoassay reagent composition of the present disclosure in a container suitable for use with an analyzer. In some embodiments, the kit comprises one or more of a Lite Reagent, a Solid Phase Reagent, or a reagent comprising a preamplifier oligomer. In various implementations, the kit may comprise one or more reagents capable of triggering chemiluminescence (e.g., Acid Reagent, Base Reagent).

[0039] These and other aspects of the invention will be better understood by reference to the following detailed description including the appended claims. BRIEF DESCRIPTION OF FIGURES

[0040] FIG. 1A provides a schematic for a sandwich immunoassay of a target involving chemiluminescent acridinium.

[0041] FIG. IB provides a schematic for a sandwich immunoassay of a target using branched DNA with chemiluminescent probe oligos.

[0042] FIG. 2 provides schematics for three alkaline phosphatase assays assessed: assay 1) provides an assay using only an alkaline phosphatase label probe; assay 2) provides an assay using multiple alkaline phosphatase label probes conjugated to an amplifier oligo; and assay 3) provides an assay using several alkaline phosphatase label probes conjugated to multiple amplifier oligos which are conjugated to a pre-amplifier oligo.

[0043] FIG. 3 provides an exemplary schematic for the assay performed in Example 3.

[0044] FIGs. 4A and 4B provide the measured relative light units in a comparative assay testing 2 component hybridizaiton schemese (“2-com”), 3 component hybridizaiton schemes (“3-com”) and a 3-component hybridization scheme without the amplifier oligomer (“3-com-Amp). FIG. 4B provides the same data as FIG. 4A in a low measurement range. Due to the assay format involving direct conjugation of the preamplifier to the solid phase (as opposed to conjugating through an analyte), the preamplifier oligo concentration is a proxy for analyte concentration. DETAILED DESCRIPTION

[0045] For convenience, certain terms employed in the specification, including the examples and appended claims, are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0046] Unless otherwise explicitly defined, the following terms and phrases are intended to have the following meanings throughout this disclosure:

[0047] All percentages given herein refer to the weight percentages of a particular component relative to the entire composition, including the carrier, unless otherwise indicated. It will be understood that the sum of all weight % of individual components within a composition will not exceed 100%.

[0048] The terms "a” or “an,” as used in herein means one or more. As used herein, the term “consisting essentially of’ is intended to limit the invention to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention, as understood from a reading of this specification. Recitations of “comprising” include “consisting essentially” and “consisting.”

[0049] The following definitions of various groups or substituents are used, unless otherwise described. Specific and general values listed below for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents. Unless otherwise indicated, alkyl, alkenyl, alkynyl, alkoxy, and the like denote straight, branched, and cyclic groups, as well as any combination thereof.

[0050] The term hydrocarbon may refer to a radical or group containing carbon and hydrogen atoms which may be bound at an indicated position (e.g., R, R', R”, RN, Y, Y’, Q. Li, Lc, RL, Rc. Ri, R2, R2a. R2b, R2c, Rs, R4, Rs, Re. R7). Examples of hydrocarbon radicals include, without limitation, alkyd, alkenyl, alkynyl, aryl, aryl-alkyl, alkyl-ary 1, and any combination thereof (e.g., alkyl-ary 1-alky l). As used herein, unless otherwise indicated, hydrocarbons may7 be monovalent or multivalent (e.g.. divalent, trivalent) hydrocarbon radicals. A radical of the form -(CH2)n-, including a methylene radical, i.e., -CH2-, is regarded as an alkyl radical if it does not have unsaturated bonds between carbon atoms. Unless otherwise specified, all hydrocarbon radicals (including substituted and unsubstituted alkyl, alkenyl, alkynyl, ary 1, aryl-alkyl, alkyl-ary1) may have from 1-35 carbon atoms. In other embodiments, hydrocarbons will have from 1-20 or from 1-12 or from 1-8 or from 1-6 or from 1-3 carbon atoms, including for example, embodiments having one, two, three, four, five, six, seven, eight, nine, or ten carbon atoms. Hydrocarbons may have from 2 to 70 atoms or from 4 to 40 atoms or from 4 to 20 atoms.

[0051] A substituted hydrocarbon may have as a substituent one or more hydrocarbon radicals, substituted hydrocarbon radicals, or may comprise one or more heteroatoms. Any hydrocarbon substituents disclosed herein (e.g., R, R’, R”, RN, Y, Y’, Q, Li, Lc, RL, Rc, Ri, R2, R2a, R2b, R2c, Rs, R4, Rs, Re, R7) may optionally include from 1-20 (e.g., 1-10, 1-5) heteroatoms. Examples of substituted hydrocarbon radicals include, without limitation, heterocycles, such as heteroaryls. Unless otherwise specified, a hydrocarbon substituted with one or more heteroatoms will comprise from 1-20 heteroatoms. In other embodiments, a hydrocarbon substituted with one or more heteroatoms will comprise from 1-12 or from 1-8 or from 1-6 or from 1-4 or from 1-3 or from 1-2 heteroatoms. Examples of heteroatoms include, but are not limited to, oxygen, nitrogen, sulfur, phosphorous, halogen (e.g.. F, Cl, Br, I), boron, or silicon. In some embodiments, heteroatoms will be selected from the group consisting of oxygen, nitrogen, sulfur, phosphorous, and halogen (e.g., F, Cl, Br, I). In certain embodiments, the heteroatoms may be selected from O, N, or S. In some embodiments, a heteroatom or group may substitute a carbon. In some embodiments, a heteroatom or group may substitute a hydrogen. In some embodiments, a substituted hydrocarbon may comprise one or more heteroatoms in the backbone or chain of the molecule (e.g., interposed between two carbon atoms, as in ’‘oxa”). In some embodiments, a substituted hydrocarbon may comprise one or more heteroatoms pendant from the backbone or chain of the molecule (e.g., covalently bound to a carbon atom in the chain or backbone, as in “oxo"’).

[0052] When an indicated group is substituted with an indicated substituent, the specified group may be substituted with one or more of any or all of the named substituents. For example, where a group, such as an alkyl or heteroaryl group, is substituted with an unsubstituted C1-C20 alkyl, or unsubstituted 2 to 20 membered heteroalkyl, the group may contain one or more unsubstituted C1-C20 alkyls, and / or one or more unsubstituted 2 to 20 membered heteroalkyls. Moreover, where a moiety is substituted with an R substituent, the group may be referred to as “R-substituted.” Where a moiety is R-substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different. If an indicated group is used multiple times in chemical genus (e g., R groups), it will be understood that each group is independently selected at each occurrence.

[0053] Unless otherwise specified, any compound disclosed herein which has one or more chiral centers may be in the form of a racemic mixture with respect to each chiral center, or may exist as pure or substantially pure (e.g.. great than 98% ee) R or S enantiomers with respect to each chiral center, or may exist as mixtures of R and S enantiomers with respect to each chiral center, wherein the mixture comprises an enantiomeric excess of one or the other configurations, for example an enantiomeric excess (of R or S) of more than 60% or more than 70% or more than 80% or more than 90%, or more than 95%, or more than 98%, or more than 99% enantiomeric excess. In some embodiments, any chiral center may be in the “S’" or “R” configurations.

[0054] It will be understood that the description of compounds herein is limited by principles of chemical bonding. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding such as regard to valencies, and to give compounds which are not inherently unstable. For example, any carbon atom will be bonded to two, three, or four other atoms, consistent with the four valence electrons of carbon.

[0055] Substituent (radical) prefix names may be derived from the parent hydride by either (i) replacing the “ane” or in the parent hydride with the suffixes “yl,” “diyl,” “triyl,” “tetrayl;” or (ii) replacing the “e” in the parent hydride with the suffixes “yl,” “diyl,” “triyl,” “tetrayl,” (here the atom(s) with the free valence, when specified, is (are) given numbers as low as is consistent with any established numbering of the parent hydride). Accepted contracted names, e.g, adamantyl, naphthyl, anthryl, phenanthryl, furyl, pyridyl, isoquinolyl, quinolyl, and piperidyl, and trivial names, e.g., vinyl, allyl, phenyl, and thienyl are also used herein throughout.

[0056] Alkyl groups typically refer to a saturated hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, Ci-Ce alkyl indicates that the group may have from 1 to 6 (inclusive) carbon atoms in it. Any atom can be optionally substituted, e.g., by one or more substituents. Examples of alkyl groups include without limitation methyl, ethyl, w-propyl, iso propyl, and / e / V-butyl. Any alkyl group referenced herein (e.g., R, R’, R”, RN, Y, Y’, Q, Li, Lc, RL, Rc, Ri, R2, R2a, R2b, R2c, R3, R4, Rs, Re, R7) may have from 1-35 carbon atoms. In other embodiments, alkyl groups will have from 1-20 or from 1-12 or from 1-8 or from 1-6 or from 1-3 carbon atoms, including for example, embodiments having one, two, three, four, five. six. seven, eight, nine, or ten carbon atoms. Alkyl groups may be lower alkyl (e.g., C1-C4 alkyl).

[0057] Haloalkyl groups are typically alkyd groups where at least one hydrogen atom is replaced by halo. In some embodiments, more than one hydrogen atom (e.g., 2. 3, 4, 5. 6, 7, 8, 9, 10, 11, 12, 13, or 14) are replaced by halo. In these embodiments, the hydrogen atoms can each be replaced by the same halogen (e.g, fluoro) or the hydrogen atoms can be replaced by a combination of different halogens (e g., fluoro and chloro). Haloalky l may include alky l moieties in which all hydrogens have been replaced by halo (sometimes referred to herein as perhaloalkyl, e.g., perfluoroalkyl, such as trifluoromethyl). Haloalky 1 groups may be optionally substituted.

[0058] Typically, alkoxy groups have the formula -O(alkyl). Alkoxy can be, for example, methoxy (-OCH3), ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 2-pentoxy, 3-pentoxy, or hexyloxy. Likewise, the term “thioalkoxy” refers to a group of formula -S(alkyl). Finally, the terms “haloalkoxy” and “halothioalkoxy” refer to -O(haloalkyl) and -S(haloalkyl), respectively. The term “sulfhydryl” refers to -SH. As used herein, the term “hydroxyl,” employed alone or in combination with other terms, refers to a group of formula -OH. Any alkoxy, thioalkoxy, or haloalkoxy group referenced herein (e.g, R, R’, R”, RN, Y, Y’, Q, Li, Lc, Rl, Rc, Ri, R2, R2a, R2b, R2c, R3, R4, Rs, Re, R7) may have from 1-35 carbon atoms. In other embodiments, alkoxy, thioalkoxy, or haloalkoxy groups will have from 1-20 or from 1-12 or from 1-8 or from 1-6 or from 1-3 carbon atoms, including for example, embodiments having one, two, three, four, five, six, seven, eight, nine, or ten carbon atoms. Alkoxy groups may be lower alkoxy (e.g.. C1-C4 alkoxy).

[0059] Aralkyl groups typically refers to groups where an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. One of the carbons of the alkyl moiety serves as the point of attachment of the aralkyl group to another moiety. Any ring or chain atom can be optionally substituted, e.g, by one or more substituents. Non-limiting examples of aralkyl include benzyl, 2-phenylethyl, and 3-phenylpropyl groups.

[0060] The term “alkenyl” may refer to a straight or branched hydrocarbon chain containing the indicated number of carbon atoms and having one or more carbon-carbon double bonds. Any atom can be optionally substituted, e.g., by one or more substituents. Alkenyl groups can include, e.g., vinyl, allyl, 1-butenyl, and 2-hexenyl. One of the double bond carbons can optionally be the point of attachment of the alkenyl substituent. Any alkenyl group referenced herein (e.g, R, R’, R”, RN, Y, Y’, Q, Li, Lc, RL, Rc, Ri, R2, R2a, R2b, R2c, R3, R4, Rs, Rs, R7) may have from 1-35 carbon atoms. In other embodiments, alkenyl groups will have from 1-20 or from 1-12 or from 1-8 or from 1-6 or from 1-3 carbon atoms, including for example, embodiments having one, two, three, four, five, six, seven, eight, nine, or ten carbon atoms.

[0061] The term alkynyl may refer to a straight or branched hydrocarbon chain containing the indicated number of carbon atoms and having one or more carbon-carbon triple bonds. Alkynyl groups (e.g, R, R’, R”, RN, Y, Y’, Q. Li, Lc, RL, Rc, Ri, R2, R2a, R2b, R2c, R3, R4, Rs, Rs, R7) can be optionally substituted, e.g., by one or more substituents. Alkynyl groups can include, e.g, ethynyk propargyl, and 3-hexynyl. One of the triple bond carbons can optionally be the point of attachment of the alkynyl substituent.

[0062] The term heterocyclyl typically refers to a fully saturated, partially saturated, or aromatic monocyclic, bicyclic, tricyclic, or other polycyclic ring system having one or more constituent heteroatom ring atoms independently selected from O, N (it is understood that one or two additional groups (e.g, RN) may be present to complete the nitrogen valence and / or form a salt), or S. The heteroatom or ring carbon can be the point of attachment of the heterocyclyl substituent to another moiety. Any atom can be optionally substituted, e.g.. with one or more substituents (e.g. heteroatoms or substituent groups X). Heterocyclyl groups can include, e.g., tetrahydrofuryl, tetrahydropyranyl, piperidyl (piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl. By way of example, the phrase “heterocyclic ring containing from 5-6 ring atoms, wherein from 1-2 of the ring atoms is independently selected from N, NH, N(Ci-Ce alkyl), NC(O)(Ci-C6 alkyl), 0, and S; and wherein said heterocyclic ring is optionally substituted with from 1-3 independently selected R” would include (but not be limited to) tetrahydrofuryl, tetrahydropyranyl, piperidyl (piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl.

[0063] The term heterocycloalkenyl typically refers to partially unsaturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon groups having one or more (e.g, 1-4) heteroatom ring atoms independently selected from 0, N (it is understood that one or two additional groups may be present to complete the nitrogen valence and / or form a salt), or S. A ring carbon (e.g.. saturated or unsaturated) or heteroatom can be the point of attachment of the heterocycloalkenyl substituent. Any atom can be optionally substituted, e.g., by one or more substituents. Heterocycloalkenyl groups can include, e g., dihydropyridyl, tetrahydropyridyl, dihydropyranyl, 4,5-dihydrooxazolyl, 4,5-dihydro-lH-imidazolyl, 1,2,5,6-tetrahydro-pyrimidinyl, and 5.6-dihydro-2H-[l,3]oxazinyl.

[0064] Cycloalkyl groups may be fully saturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon groups. Any atom can be optionally substituted, e.g., by one or more substituents. A ring carbon serves as the point of attachment of a cycloalkyl group to another moiety. Cycloalkyl moieties can include, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbomyl (bicyclo[2.2.1]heptyl).

[0065] Cycloalkenyl groups may be partially unsaturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon groups. A ring carbon (e.g., saturated or unsaturated) is the point of attachment of the cycloalkenyl substituent. Any atom can be optionally substituted, e.g., by one or more substituents. Cycloalkenyl moieties can include, e.g., cyclohexenyl, cyclohexadienyl, or norbomenyl.

[0066] Aryl groups are often aromatic monocyclic, bicyclic (2 fused rings), tricyclic (3 fused rings), or polycyclic (> 3 fused rings) hydrocarbon ring system. One or more ring atoms can be optionally substituted, e.g., by one or more substituents. Aryl moieties include, e.g., phenyl and naphthyl.

[0067] Heteroaryl groups typically are aromatic monocyclic, bicyclic (2 fused rings), tricyclic (3 fused rings), or polycyclic (> 3 fused rings) hydrocarbon groups having one or more heteroatom ring atoms independently selected from O, N (it is understood that one or two additional groups may be present to complete the nitrogen valence and / or form a salt), or S in the ring. One or more ring atoms can be optionally substituted, e.g.. by one or more substituents. Examples of heteroaryl groups include, but are not limited to, 2H-pyrrolyL 3H-indolyl, 4H-quinolizinyl, acridinyl, benzo[b]thienyl, benzothiazolyl, P-carbolinyl, carbazolyl, coumarinyl, chromenyl, cinnolinyl, dibenzo[b,d]furanyl, furazanyl, fury l, imidazolyl, imidizolyl, indazolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl. naphthyridinyl, oxazolyl. perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl. phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl. thiadiazolyl, thianthrenyl, thiazoly l, thienyl, triazolyl, and xanthenyl.

[0068] In general, when a definition for a particular variable includes both hydrogen and nonhydrogen (halo, alky l, aryl) possibilities, the term “substituent(s) other than hydrogen” refers collectively to the non-hydrogen possibilities for that particular variable, unless otherwise specified.

[0069] In general, the limits (end points) of any range recited herein are within the scope of the invention and should be understood to be disclosed embodiments. Additionally, any halfintegral value within that range is also contemplated. For example, a range of from 0 to 4 expressly discloses 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, and any subset within that range (e.g., from 1 to 2.5).

[0070] The term “substituent” may refer to a group “substituted” on, on a hydrocarbon (e.g, an alkyl, haloalkyl, cycloalkyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, heteroaryl) group at any atom of that group, typically replacing one or more hydrogen atoms therein. In one aspect, the substituent(s) on a group (e.g., R, R’, R”, RN, Y, Y’, Q, Li, Lc, RL, Rc, Ri, R2, R2a. R2b, R2c, Rs, R4, Rs, Re, R?) are independently any one single, or any combination of two or more of the permissible atoms or groups of atoms delineated for that substituent. In another aspect, a substituent may itself be substituted with any one of the above substituents. In some embodiments, an indicated substituent is not further substituted. Further, as used herein, the phrase “optionally substituted” means unsubstituted (e.g., substituted with an H) or substituted. It is understood that substitution at a given atom is limited by valency. Common substituents include halo (e.g. F), C1-12 straight chain or branched chain alkyl, C2-12 alkenyl, C2-12 alkynyl, C3-12 cycloalkyl, Ce-12 aryl, C3-12 heteroaryl, C3-12 heterocyclyl, C1-12 alkylsulfonyl, nitro, cyano. -COOR, -C(0)NRR’, -OR. -SR, -NRR', and oxo, such as mono-or di- or tri-substitutions with moieties such as trifluoromethoxy, chlorine, bromine, fluorine, methyl, methoxy, pyridyl, furyl, triazyl, piperazinyl, pyrazoyl, imidazoyl, and the like, each optionally containing one or more heteroatoms such as halo, N, 0, S, and P. R and R’ are independently hydrogen, C1-12 alkyl, C1-12 haloalkyl, C2-12 alkenyl, C2-12 alkynyl, C3-12 cycloalkyd, C4-24 cycloalkylalkyl, C6-12 aryl, C7-24 aralkyl, C3-12 heterocyclyl, C3-24 heterocyclylalkyl, C3-12 heteroaryl, or C4-24 heteroarylalkyl. Unless otherwise noted, all groups described herein optionally contain one or more common substituents, to the extent permitted by valency. The term “substituted” typically means that a hydrogen and / or carbon atom is removed and replaced by a substituent (e.g, a common substituent). The use of a substituent (radical) prefix names such as alkyl without the modifier “optionally substituted” or “substituted” is understood to mean that the particular substituent is unsubstituted. However, the use of “haloalky l” without the modifier “optionally substituted” or “substituted” is still understood to mean an alkyl group, in which at least one hydrogen atom is replaced by halo and any other associated substitutions as necessary. Any hydrocarbon described herein may be considered optionally substituted.

[0071] The analyte may be expressed on a cancer cell surface and / or be more highly expressed in cancer cells than in normal cells, or an antigen that is known to be specifically expressed only in cancer cells. Examples of such an antigen include, but are not limited to, cell-surface vimentin (CSV), epithelial cell adhesion molecule (EpCAM, CD326), programmed cell death ligand-1 (PD-L1), glypican-3 (GPC3), glypican-1 (GPC1), carcinoembryonic antigen (CEA), CD4, CD5. CD16, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD52, CD123, CD171, Cancer Antigen 125. Claudinl8. receptor-type tyrosine kinase R0R1. sialyl. Le.sup.x-i (SLX), Sialyl Lewis A (CAI9-9), immune co-stimulatory protein B7-homolog6 (B7H6), cell surface glycoprotein (CS-1), receptor-type tyrosine kinase 3 (FLT3), prostate-specific membrane antigen (PSMA), B-cell maturation antigen (BCMA), Wilms tumor 1 (WT1), NY-ESO-1, Caveolin-1, Fas ligand (FasL), TNF-related apoptosis-inducing ligand (TRAIL), Galectine3, CD 151, Tetraspanin 8, epithelial growth factor receptor (EGFR), HER2, Ribophorin 2 (RPN2), CD44, and TGF-p. In various implementations, the protein may be Adiponectin, Alpha 1 Antitrypsin, Alpha 2 Macroglobulin, Angiopoietin 1, Angiotensin Converting Enzy me. Apolipoprotein(a), Apoliprotein AI, Apolipoprotein All, Apolipoprotein B, Apolipoprotein CI, Apoliproprotein CIII, Apolipoprotien H, Beta 2 Microglobulin, Brain Derived Neurotrophic Factor, C Reactive Protein, Calbindin, Carbonic anhydrase 9. Carcinoembryonic antigen related cell adhesion molecule I, CD5 Antigen like. Cystatin. Cecorin, E Selectin, ENRAGE, Eotaxin 1, Factor VII, Fatty Acid Binding Protein, Ferritin, Fetuin A, Fibrinogen, Follicle Stimulating Hormone Glucagon-like Peptide-1, Granulocyte Macrophage, Colony Stimulating Factor Growth Hormone, Haptoglobin, Immunoglobulin A, Immunoglobulin M, Insulin, Intercellular Adhesion Molecule-1, Interferon gamma,, Interferon gamma Induced Protein 10, Interleukin-1 alpha. Interleukin-1 beta, lnterleukin-1 receptor antagonist, lnterleukin-2. Interleukin-3, Interleukin-4, Interleukin-5, Interleukin-6, Interleukin-7, Interleukin-8, Interleukin-10, Interleukin-12 Subunit p40, Interleukin-12 Subunit p70, Interleukin-15, Interleukin-17, Interleukin-18. Interleukin-18 binding protein, Interleukin-23, Kidney Injury Molecule 1, Lectin Like Oxidized LDL Receptor 1, Leptin. Lipoprotein(a), Lutenizing Hormone, Macrophage Inflammatory Protein 1 alpha, Macrophage Inflammatory Protein 1 beta, Macrophage Inflammatory Protein 3 alpha, Matrix Metalloproteinase 1, Matrix Metalloproteinase 2, Matrix Metalloproteinase 3, Matrix Metalloproteinase 7, Matrix Metalloproteinase 9, Matrix Metalloproteinase 9 Total, Matrix Metalloproteinase 10, Midkine, Monocyte Chemotactic Protein 1, Monocyte Chemotactic Protein 2, Monocyte Chemotactic Protein 4, Monokine Induced by Gamma Interferon, Myeloid Progenitor Inhibitory Factor 1, Myeloperoxidase, Myoglobin, N terminal prohormone of brain natiuretic peptide, Osteopontin, Pancreateic Polypeptide, Plasminogen Activator Inhibitor 1, Platelet endothelial cell adhesion molecule, Prolactin, Pulmonary and Activation regulated Chemokine, Pulmonary surfactant-assocaited protein D, Resistin, Serotransferrin, Serum Amyloid P Comoponent, Stem Cell Factor, T-Cell Specific Protein RANTES, Tamm Horsfall Urinary7 Glycoprotein, Thrombomodulin, Thrombospondin 1, Thyroid Stimualting Hormone, Thyroxine Binding Globulin, Tissue Inhibitor of Metalloproteinases, Transthyretin. Troponin, Tumor Necrosis Factor alpha, Tumor Necrosis Factor beta, Tumor Necrosis Factor receptor 2, Vascular Cell Adhesion Molecule 1, Vascular Endothelial Growth Factor, Vitamin D Binding Protein, Vitamin K-Dependent Protein S, Vitronectin, von Willebrand Factor.

[0072] Another embodiment may be directed to non-limiting examples of diseases that may be tested for biomarkers or disease state targets include: thyroid disease, including hyperthyroidism, hypothyroidism, autoimmune disorders, such as Hashimoto's disease or Graves' disease (e.g., thyroid peroxidase (TPO), Tg, Free T3, Free T4, Total T3, Total T4, TsH3-ultra, TSH, T uptake), hepatitis (e.g., HBe, HBs 2, HAV IgM, HAV Total, HBc IgM, HBc Total, HBeAg. HBsAg confirmatory, HBsAgll, HBsAgll Quant, HCV), HIV (e.g., HIV 1 / 0 / 2 Enhanced (EHIV), HIV Combo (CHIV)), cancers (e g., AFP, BR 27.29, CA 125II, CA 15-3, CA 19-9, Calcitonin, CEA, Complexed PSA, Free PSA, PSA, Serum HER-2 / neu), prostate cancer (e.g., prostate-specific antigen (PSA)), ovarian cancer (e.g., CA125), squamous cell carcinoma of the head and neck (SCCHN) (e.g., macrophage inflammatory protein lb (MIP lb), interleukin 13 (IL 13), metalloproteinase 3 (MMP3), epidermal growth factor (EGF) vascular cell adhesion molecule (VCAM), squamous cell carcinoma antigen (SCC-Ag) and neurite growth-promoting factor 2 (Midkine)), and squamous cell carcinoma of the tongue (SCCT) (e.g., NT-3, TNFB, CDS, uPA, IL-lra, Flt3L, DNER, CXCL1, CD6, CD40, CDH3, PECAM-1, TIE2, FasL, FR-alpha, CD69, CD244, TWEAK, IL-12B, EGFR, NTRK3, ErbB4 / HER4, IL-12), diabetes (e.g., C-Peptide. insulin), Kidney Risk Inflammatory Signature (KRIS) which may indicate the risk of progression of diabetic kidney disease to end stage renal disease (e.g., TNF-RSF1A, TNF-RSF1B, TNF-RSF21, TNF-RSF19, TNF-RSF27, TNF-RSF19L, IL-15RA, IL-17F, CD55, CD300C, TNF-SF15, CCL14, CCL15, CSF1, HAVCR2, IL-1R1, IL-18R1). cardiovascular disease, such as, Atherosclerotic Cardiovascular Disease (ASCVD), heart failure, etc. (e.g., growth differentiation factor 15 (GDF15), tissue inhibitor of metalloproteinase-1 (TIMP1), beta-2-microglobulin (B2M), NT-proBNP, adrenomedullin (ADM), C-type lectin domain family 3 member B (CLEC3B), insulin-like growth factor 1 (IGF1), butyrylcholinesterase (BCHE), paraoxonase 1 (PON1), insulin-like growth factor binding protein 1 (IGFBP1), IGFBP2, IGFBP3, CNTN1, kallikrein Bl (KLKB1), and peripheral myelin protein 2 (PMP2), REGIA, Cystatin-C, d troponin, AGP1, sICAMl, CRP, CDSL, CD14, MPO, UCMGP, EFEMP1, GRN, Adipsin, Resistin, AIM, d IL6, FGF23, MMP8, MMP9, SAA1, BCHE, PON1, MCP1, sGP130, Ceruloplasmin, COL18A1. NRCAM, CDH13, LDLR, d_FLT3. FBN, d_CSF2RB, p-adrenergic receptors (PARs) (norepinephrine endogenous ligand), Angiotensin II type 1 receptors (ATIRs) (Angll endogenous ligand), Alzheimer's disease and / or mild cognitive impairment (e.g., 0NECUT2, SIRT1, BCL2, PSEN1), tumor angiogenesis (e.g., delta-like 4 (DLL4)), etc. Some of these biomarkers or targets may be increased or decreased in a subject suffering from a disease or condition associated with the marker or target being tested. The methods may include the evaluation or monitoring of the progression of the disease state using the immunoassays of the present deisclosure. Importantly, the methods of detection described here are directed to the improved sensitivity and accuracy of detection of targets or biomarkers that may not be easily detected and may provide more facile evaluation or monitoring as compared to detection methodologies that do not use branched DNA (e.g., acridinium based immunoassays without the branched DNA oligomers).

[0073] In various embodiments, the biomarkers may include, but are not limited to, neural proteins such as Microtubule Associated Protein Tau (Tau, total protein and / or phosphorylated fraction). Amyloid-beta 42 (Ap42), Neuroligin (NLGN, including in particular NLGN1), TAR DNA-binding protein 43 (TDP43), neurogramn (NRGN), SYP, Cathepsin D, LC3, SYT, BIM, NEFL, ENO2, GPR26 and combinations thereof, as discussed in further detail and exemplified below. In another embodiment the one or more biomarkers are selected from the group consisting of Tau, p-Tau, Ap42, and NLGN. TDP43, CLU, SYP, BIM, NEFL, ENO2, NRGN, and GPR26 gene products, and combinations thereof. Each possibility represents a separate embodiment of the invention. In a particular embodiment, phosphorylated Tau includes Thr-181-phosphorylated Tau (pl81-Tau). In some embodiments, the biomarker is selected from the group consisting of: LC3, Cathepsin D, NRF2, Ap42, p-Tau, PSD95, proBDNF, COX2, EIF2C2 and NF-kB. In other embodiments, the biomarkers may be selected from the group consisting of: total-Tau, p-Tau, AP42, PSD95, proBDNF and NFkB. In other embodiments, the biomarkers may include total-Tau, p-Tau, Ap42, PSD95, proBDNF and NFkB. In another embodiment said biomarkers are Ap42, p-Tau, PSD95 and proBDNF. In another embodiment the biomarker is selected from the group consisting of LC3, TDP43, NRF2 (NFE2L2), cathepsin D. COX2 and EIF2C2. In yet other embodiments, the biomarker may be selected from the group consisting of LC3, TDP43, and NRF2. In other embodiments, the biomarkers may include LC3, TDP43, and NRF2. The analyte may be Tau, total-Tau, p-Tau, Neuroligin 1, Neurogranin, Synaptophysin, 5-HT2A, TDP-43, A9Beta)40, Ap42, CTFp, LC3B, LC3, Cathepsin D. NRF2. BAD, p-BAD. PSD95, proBDNF, mature BDNF, COX2, EIF2c2, NFkB, LAMP-2, Clusterin, BIM, NFL, Enolase 2, or GPR26.

[0074] In certain implementations, the labeled probe oligomer is conjugated to one or more primer sets that may be used to amplify a gene (or specific region thereof). For example, the probe oligomer may be conjugated a primer set for the V3 region of the HIV ev gene.

[0075] The present disclosure involves conjugates useful in high sensitive immunoassays for the detection of an analyte. Typically, these conjugates have a chemiluminescent acridinium conjugated to a probe oligo via a linker (e.g., PEG such as PEG2-PEG20 or PEG3-PEG10).

[0076] In some embodiments, the chemiluminescent acridinium is conjugated to the probe oligo by reacting the oligomer with a compound comprising a reactive functional group. For example, the compound may have the structure: RFG-L-T (I) wherein RFG is a reactive functional group for conjugation (e.g., to an oligomer), L is absent (i.e., it is a bond) or a linker optionally comprising a group Lc or ZL, and T is a chemiluminescent acridinium comprising the structure: and “k” are independently 0 (e.g., all R2 groups are hydrogen, all R3 groups are hydrogen), 1, 2, 3, or 4; Ri is hydrogen, -R, -Xb, -RL-Xb, -Lc-R, -Lc-Xb (e.g., -Li-Xb), -Z, -RL-Z, -Lc-Z (e.g., -Li-Z), or -Rl-Lc-Rl-Z (e.g., -Rl-Li-Rl-Z); R2 and R3 are independently selected at each occurrence from hydrogen, -R, an electron donating group, -Xc, -RL-XC, -Lc-Xc (e.g., -Li-Xc), and -Z; wherein two vicinal R2 or R3 groups may together form a fused cyclic group (e.g.. 5-7 membered fused and or heteroaryl group, 5-7 membered fused heterocyclic group) and wherein R2 or R3 may comprise a linkage to an imaging agent such as a fluorophore (e.g., rhodamine); Lc is a divalent C1-35 alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted (e.g.. with 1 to 20 heteroatoms, with 1-20 substituents); ZL is a zwitterionic linker group having the structure: “m” is 0 (i.e. it is a bond) or 1; and “p” are independently at each occurrence an integer from 0 (i.e. it is a bond) to 10: Z is a zwitterionic group independently at each occurrence has the structure: and “F are independently 0 or 1; “r” is independently an integer from 0 to 10 (e.g., from 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); Xa and Xb are independently at each occurrence an anionic group; Xc is a protonated anionic group; Li is independently at each occurrence -0-, -S-, -NH-, -N(Rn)-, -(CH2)i-io-, -S(=O)i-2-, -C=C- -C=C-(CH2)i-3- -C(O)- -O-C(O)-, -C(O)-(CH2) 1-4-, -(CH2) 1-4-0(0)-, -C(O)-O-, -C(0)-N(Rn)-, -C(0)-NH-, -N(Rn)-C(0)-, -NH-C(O)-, -C(O)-N(Rn)-(CH2)i-3-, -(CH2)i-3-C(O)-N(Rn)- -(CH2)i-3-N(Rn)-C(O)-, -NH-S(O)i-2-, -N(Rn)-S(0)1-2-, -S(0)i-2-N(Rn)-, -S(O)i-2-NH-, -(CH2)i-3-NH-S(0)i-2-, -(CH2)i-3-N(Rn)-S(O)i-2-, -(CH2)i-3-S(O)i-2-N(Rn)- -(CH2)i-3-S(O)i-2-NH-, -O-(CH2)i-4-, -(CH2)i-4-O-, -S-(CH2)i-4-, -(CH2)i-4-S-, -NH-(CH2)i-4-. -N(Rn)-(CH2) 1-4-, -(CH2)i-4-N(Rn)- -(OCH2)i-io-, -(CH20)i-io-, -(OCH2CH2)i-io-, or -(CH2CH20)i-io-; Rl is independently at each occurrence a C1-20 bivalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, phenyl, mono alkyl substituted phenyl, di alkyl substituted phenyl, alkynyl. arylalkyl), optionally having one or more (e.g, 1-10, 1-5) points of substitution (e.g, with 1-10 heteroatoms, with 1-10 substituents); R is independently at each occurrence hydrogen or C1-35 hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms, with 1-20 substituents); R' and R" are independently at each occurrence hydrogen or a C1-10 alkyl; Rn is independently at each occurrence from hydrogen or C1-5 alkyl (e.g, methyl, ethyl, propyl); and R’ is hydrogen or a Ci-io alkyl; or a salt thereof (e.g., a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxylate salt). For example, the chemiluminescent acridinium comprising a reactive functional group has the structure of formula (la): Ri I L I RFG (la) wherein is 0 or N; Y is selected from -R or -RL-Z, or in the case where Q is 0 then Y is absent; and Y’ is either absent (i.e. it is a bond), or is selected from -Li-, -RL-, -Rl-Li-, -Li-Li-, -Li-Rl-, -Li-Rl-Li, and -Rl-Li-Rl-. In some embodiments, the chemiluminescent acridinium comprising a reactive functional group has the structure of formula (la) or (lb): Ri l L I RFG (lb) Ri I I o=s=o I Y" L I RfG wherein R4-R7 are independently hydrogen, an electron donating group, or C1-35 alkyl, alkenyl, alkynyl, and, alkoxy, alkylthio, or amino; and Y” is either absent (i.e., it is abond) or-Lc- -Li-, -RL-, or -Rl-Li- In some embodiments. L is absent or C1-C5 alkylene. In particular embodiments, the reactive functional group is an N-succinimidyl ester. In various implementations, at least one of R2 and R3 (e.g., at the C2 and / or C7 positions of the acridinium) are independently -Xc. -R1 -Xc, -Lc-Xc (e.g., -Li-Xc). In some embodiments, at least one of R2 and R3 are independently alkoxy (e.g., C1-C4 alkoxy) substituted with -C(O)OH, -SO2OH), -OSO2OH), -0P(0)(0Rp)0H, -OH, or combinations thereof.

[0077] When a moiety of the compounds of the present disclosure are described as comprising an analyte or a binding partner thereof or oligomer in the context of molecular conjugates, a covalent linkage may be formed with an analyte or binding partner thereof (e.g., using the reactive functional groups which form covalent linkages) and the remaining portions of the conjugate. In such conjugation, the analyte or binding partner thereof may have a hydrogen on the unconjugated analyte or binding partner thereof that forms a covalent bond to the indicated moiety. Conjugation to the oliogopeptides may occur, for example, by 3’-terminal modification or 5-terminal modification. For example, oligo conjugation may occur through the formation of disulfide bonds betw een oligo (and analyte or linked analyte) or oligo and chemiluminescent moiety with reactive functional groups and linkers as described herein.

[0078] The 3' and 5' ends of an oligonucleotide can be modified. Such modifications can be at the 3' end, 5' end or both ends of the molecule. They can include modification or replacement of an entire terminal phosphate or of one or more of the atoms of the phosphate group. For example, the 3' and 5' ends of an oligonucleotide can be conjugated to other functional molecular entities such as Ithe chemiluminescent moiety or the antibody or fragment thereof for conjugation to the analyte. These functional molecular entities may be attached to the sugar through a phosphate group and / or a linker. The terminal atom of the spacer can connect to or replace the linking atom of the phosphate group or the C-3' or C-5' 0, N, S or C group of the sugar. Alternatively, the spacer can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs). These spacers or linkers can include e.g., -(CH2)n-, -(CH2)nN— (CH2)nO-, -(CH2)nS-, (e.g., n=l to 8 or 3 to 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents.

[0079] In some embodiments, any hydrocarbon or substituted hydrocarbon disclosed herein (e.g. R, R’, R”, Rn, Y, Y’, Q, Li, Lc, Rl, Rc. Ri. R2, R2a. R2b, R2c. Rs, R4, Rs, Re, R?) may be substituted with one or more (e.g., from 1-6 or from 1-4 or from 1-3 or one or two or three) substituents X, where X is independently selected at each occurrence from one or more (e.g, 1-20) heteroatoms or one or more (e.g.. 1-10) heteroatom-containing groups, or X is independently selected at each occurrence from -F, -Cl, -Br, -I, -OH, -OR*, -NH2. -NHR*, -N(R*)2, -N(R*)3+, -N(R*)-0H, -N(^0)(R*)2, -0-N(R*)2, -N(R*)-0-R*, -N(R*)-N(R*)2, -C=N-R*, -N=C(R*)2, -C=N-N(R*)2, -C(=NR*)(-N(R*)2), -C(H)(=N-OH), -SH, -SR*, -CN, -NC, -CHF2, -CC13, -CF2CI, -CFCh, -C(=O)-R*, -CHO, -CO2H, -C(O)CH3, -COT. -CO2R*, -C(=O)-S-R*, -O-(C=O)-H, -O-(C=O)-R*, -S-C(=O)-R*. -(C=O)-NH2, -C(=O)-N(R*)2. -C(=O)-NHNH2. -O-C(=O)-NHNH2, -C(=S)-NH2. -(C=S)-N(R*)2, -N(R*)-CHO, -N(R*)-C(=O)-R*, -C(=NR)-OR*, -O-C(=NR*)-R*, -SCN, -NCS, -NSO, -SSR*, N(R*) C(=O) N(R*)2, CHs, CH2 CH3, CH2 CH2 CH3, C(H)(CH2)2, C(CH3)3, -N(R*)-C(=S)-N(R*)2, -S(=O)i-2-R*, -O-S(=O)2-R*, -S(=O)2-OR*, -N(R*)-S(=O)2-R*, -S(=O)2-N(R*)2, -O-SO3, -O-S(=O)2-OR*, -O-S(=O)-OR*. -O-S(=O)-R*, -S(=O)-OR*, -S(=O)-R*, -NO, -NO2, -NO3, -O-NO, -O-NO2, -N3, -N2-R*, -N(C2H4), -Si(R*)s, -CF3, -O-CF3, -0-CHF2, -O-CH3, -O-(CH2)i-6CH3, -OC(H)(CH2)2 -OC(CH3)3, -PR*2, -o-P(=O)(OR*)2, or -P(=O)(OR*)2; where, independently at each occurrence. R* may be H or a C1-10 or C1-8 or C1-6 or Ci-4 hydrocarbon, including without limitation alkyl, alkenyl, alkynyl, aryl (e.g., phenyl), alkyl-aryl (e.g., benzyl), aryl-alkyl (e.g., tolyl) In some embodiments, X may comprise a Ci-Cs or Ci-Ce or C2-C4 perfluoroalkyd. In some embodiments, X may be a Ci-Cs or C2-C6 or C3-Cs heterocycle (e.g., heteroaryl radical). The term “halo” or “halogen” refers to any radical of fluorine, chlorine, bromine or iodine. In some embodiments. X is independently selected at each occurrence from -OH, -SH, -NH2, -N(R*)2, -C(O)OR*, -C(O)NR*R*, -C(O)NR*R*. -C(O)OH, -C(O)NH2, F, or -Cl. In some embodiments, X is F. R and R* may be, independently at each occurrence, saturated or unsaturated alkyl (e.g., Ci-Cs alkyl). In some embodiments, R and R* are independently selected from hydrogen, methyl, ethyl, propyl, or isopropyl. In some embodiments, R and R* are independently selected from hydrogen, methoxy, ethoxy, propoxy, or isopropoxy. In some embodiments, X is -CF3 or -0-CF3.

[0080] Lc may have the structure: -(Xl)o-l-(RL)o-5-(X2)o-1- (RL)o-5-(X3)o-1-(RL)o-5-(X4)o-1-(RL)o-5- wherein Xi is selected from =N-, -0-, -S-, or -NRn-; X2-X4 are independently selected from -0-, -S-, -NRn-, -C(0)-, -NRn-C(O)-, -C(0)-NRn-, -O-C(O)-, or-C(O)-O-, -S-C(O)-, or-C(O)-S-; and Rl is independently selected at each occurrence from -CH2-, -(CH2CH2O)-, or -(OCH2CH2)-. In various embodiments, Lc comprises at least one atom (or at least two atoms) in the chain between A and and the oligomer such as the preamplifer oligomer (or between the oligomer (e.g., probe oligomer) and T).

[0081] Anionic groups, such as Xa, Xb, Xc may be, for example, independently at each occurrence carboxylate (-C(O)O'), sulfonate (-SO3), sulfate (-OSO3), phosphate (-OP(O)(ORp)O), or oxide (-O'), and Rp is hydrogen or C1-12 hydrocarbon optionally having one or more (e.g.. 1-10, 1-5) points of substitution (e.g, with 1-10 heteroatoms, with 1-10 substituents). Protonated versions of these groups (which may be protonated or in salt form to satisfy charge neutrality of a compound) include -C(O)OH, -SO2OH), -OSO2OH), -OP(O)(ORp)OH, or -OH, and Rp is hydrogen or C1-12 hydrocarbon optionally having one or more (e.g, 1-10, 1-5) points of substitution (e.g, with 1-10 heteroatoms, with 1-10 substituents) For example, Ri may comprise (or be) -RL-SO3 (e.g., sulfopropyl). In some embodiments, Ri comprises (or is) sulfopropyl. In some embodiments, Ri, R2, and / or R3 comprise (or are) sulfopropyl (which may be in zwitterionic formed (e.g., Ri) or neutral (e.g., R2, R3) form). In some embodiments, Ri is -S(O)2-NH-Z or -(CH2)i-3-S(O)2-NH-Z. In various implementations, R2 and R3 are independently at each occurrence hydrogen, alkyl, or alkoxy (eg, lower alkoxy such as C1-C4 alkoxy, methoxy, ethoxy, propoxy, isopropoxy) optionally substituted with -C(O)OH, -SO2OH), -OSO2OH), -OP(O)(ORp)OH, or -OH. In some embodiments, R2 and R3 are each hydrogen. In other embodiments, one of R2 or R3 is hydrogen and the other of R2 or R3 is alkoxy (e.g, lower alkoxy such as C1-C4 alkoxy, methoxy, ethoxy, propoxy, isopropoxy). In some embodiments, Xa, Xb, or Xcis sulfonate (-SO3), m is 1, Rl is propyl, and n andp are each 3. For example, ZL may have the structure: R'

[0082] The compounds may be used to detect for the presence of a material in a sample such as an analyte (e.g., a biomolecule). In some embodiments, the analyte is a thyroid hormone (e.g., a thyroid stimulating hormone and, for example, the antibody is a binding partner therefor such as an anti-thyroid stimulating hormone monoclonal antibody (AntiTSH-mAb)), an androgen, a steroid hormone (e.g., androstenedione, testosterone), a troponin, thyroglobulin, anti-thyroid peroxidase antibody, triiodothyronine (T3) hormone, thyroxine (T4) hormone, thyroxine-binding globulin (TBG), neurofilament such as neurofilament light chain (e.g, serum neurofilament light chain), a vitamin (e.g., vitamin-D such as 25-hydroxy-vitamin D), or an antibody for a virus (e.g., hepatitis). In some embodiments, the biological sample is blood and the analyte must pass through the blood brain barrier to enter the blood (e.g., the analyte is neurofilament light chain). In these embodiments, the high sensitivity assay provided by the assays allows for even the low blood concentration levels of analytes.

[0083] In some embodiments, the compounds of the present disclosure may be zwitterionic and include one or more zwitterionic groups. For example, the Ri group attached to the positively charged nitrogen of the acridinium may optionally substituted with up to 20 heteroatoms (e.g., N, O, S, P, Cl, Br, F) and therefore may in combination with the positively charged acridinium nitrogen atom, constitute a zwitterionic group. For example, a sulfopropyl or sulfobutyl group attached to the acridinium nitrogen may form a zwitterionic pair. The Ri group may also be neutral (e.g, lower alkyl such as methyl) or by itself be zwitterionic (e.g, Ri is -Z, -Rl-Z, -Ls-Z, or -Rl-Ls-Rm-Z). In some embodiments, Ri has the structure: When the acridinium label is charged (e.g., Ri has a net neutral charge), the compound may be in its salt form and optionally include a counterion to balance the positively charged nitrogen of the acndimum nucleus. The counterion may be selected from CH3SO4; FSO3; CFsS04; C4F9SO4; CH3C6H4SO3; halide (e.g, Cl; F; Br ), CF3COO; CH3COO; or NOs'. In some embodiments, Ri is methyl, ethyl, propyl, or isopropyl. In some embodiments, the acridinium compound may be zwitterionic by via covalent attachment to an anion. For example, Ri may comprise -RL-Xa and -Xa is sulfonate (-SO3). In some embodiments. Ri is -RL-Xa and -Xa is sulfonate (-SO3). In some embodiments, Ri is -RL-X or -Ls-Z. In some embodiments, Ls is -S(O)2-NH- or -(CH2)i-3-S(O)2-NH-. Ri may comprise a sulfopropyl group (-(CH2)3-SO3). In a specific embodiment. Ri is sulfopropyl.

[0084] The substituents on the chemiluminescent acridinium ester may be modified to vary the rate and yield of light emission, to reduce the non-specific binding, increase stability; or increase hydrophilicity. Typically, these modifications will have minimal interference substantially with the binding of the analyte and its binding partner. Examples of substituent variability are disclosed in Natrajan et al. in U.S. Pat No 7,309,615, hereby incorporated by reference herein, which describes high quantum yield acridinium compounds containing electron donating groups such as alkoxy groups (OR*) at, for example, C2 and / or C7, wherein R* is a group comprising a sulfopropyl moiety or ethylene glycol moieties (e.g., -0(CH2CH20)o-5CH3) or combinations thereof. In some embodiments, R2 (e.g., R2a, R2b, R2e) and / or Rs may be independently at each occurrence hydrogen an electron donating group such as an alkoxy group (e.g., OR such as -0(CH2CH20)o-sCH3 and / or OR*.) or a group OG. Natrajan et al. in International Pub. No. WO2015 / 006174, hereby incorporated by reference in its entirety, also describes hydrophilic high quantum yield, chemiluminescent acridinium esters possessing certain electron-donating functional groups, for example, groups at the C2 and / or C7 positions as well. These electron donating groups ( OG) may have the structure: wherein R9-R14 are independently selected at each occurrence a methyl group or a group -(CH2CH2O)aCH3, where a is an integer from 1 to 5. G may be independently selected at each occurrence from, for example, hydrogen, alkyl (e.g., C1-C4 alkyl), -(CIhCIhOp-io-OCfk such as -(CH2CH2O)2-OCH3 or-(CH2CH2O)5-OCH3.

[0085] T may comprise two flanking methyl groups on a phenolic ester to stabilize the bond as disclosed in Law et al. Journal of Bioluminescence and Chemiluminescence 4: 88-89 (1989), hereby incorporated by reference in its entirety. In some embodiments T in the conjugate has the structure:

[0086] The reactive functional group may an amine-reactive group, a thiol-reactive group, a carboxy-reactive group, a maleimidyl-reactive group, or a carbohydrate-reactive group. In some embodiments, the reactive functional group may react with a functional group of the analyte or binding partner therefore such as a primary amine. The reactive functional group may comprise (or be) an isiothiocyanate, isocyantate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, and halide, imidoester, carbodiimide, anhydride, fluorophenyl ester, or combinations thereof. In various implementations, the reactive functional group labels the analyte or binding partner therefor through acylation or alkylation. For example, the linkage may be formed from a reactive functional group (RFG) selected from: phosphoramidites (e.g., -OP(OR)N(RR) wherein R is independently at each occurrence, for example, hydrogen or optionally substituted alkyl such as C1-C4 alkyl), In some embodiments, the compound comprises a linker group having the structure -NH-C(0)- or -C(0)-NH-, -0(0)0-, or -0C(0)-. In a preferred embodiment, the compound or moiety thereof (e.g., Lc, T) comprises at least one -NH-C(O)-, -C(O)-NH-, -0(0)0-, or -0C(0)- linker group.

[0087] The covalent linkage between RFG and T (e.g., L) may comprise (or be) a divalent Ci-20 alkyl, alkenyl, alkynyl, and. or arylalkyl radical, optionally substituted with up to 20 heteroatoms (e.g., N, O. S, P. CL F, Br). In some embodiments L comprises a zwitterionic linker. L may have the structure -Lc-(Zl)z-, wherein z is 0 or 1. Lc may have the structure -(Xl)o-l—(RL)0-5—(X2)o-1- (RL)o-5-(X3)o-1-(RL)o-5-(X4)o-1-(RL)o-5- wherein Xi is selected from -0-, -S-, -NRn-, -0(0)-, -NRn-C(O)-, -C(O)-NRn-, -0-C(0)-, or-0(0)-0-, -S-C(O)-, or-C(O)-S-, =N-, -0-, or-S-; X2-X4 are independently selected from -0-, -S-, -NRn-, -C(0)-, -NRn-C(O)-, -C(0)-NRn- -O-C(O)-, or-C(O)-O-, -S-C(O)-, or-C(O)-S-; and Rl is independently selected at each occurrence from-CH2-, -(CH2CH2O)-, or -(OCH2CH2)-; with, for example, the proviso that Lc comprises at least one atom (or at least two atoms) in the chain between A and T (or between A and ZL).

[0088] In some embodiments, L and / or T comprises -C(O)-NH- In some embodiments, Lc has the structure:

[0089] The detectable label may comprise a dimethyl acridinium ester (DMAE) moiety and a zwitterionic linker comprising a zwitterionic linker or a polyethylene glycol derived linker to improve properties of the compound. Such properties as non-specific binding, hydrophilicity, or compound stability may be improved when T comprises a zwitterionic linker or a polyethylene glycol derived linker or a dimethyl phenyl ester. In some embodiments, ZL has the structure: R' In several embodiments, R’ is hydrogen or lower alkyl (e.g., methyl, ethyl, propyl).

[0090] Chemiluminescence from multiple acridinium labels can be measured by using photomultiplier tubes (PMT), each of the PMT’s which may be equipped with an optical filter that allows the light from an acridinium ester of interest to pass through while blocking the unwanted light. Yet another alternative detector is a charge-coupled device (CCD). The chemiluminescence may pass through a grating, such that the wavelength separation may occur along the detector (e.g., CCD detector) and the images can be analyzed accordingly.

[0091] The acridinium labels of U.S. Pat. No. 8,119,422, which is hereby incorporated by reference in its entirety, can be used as materials to form the wavelength separated and emission separated sets of acridinium labels.

[0092] The compounds can be prepared from commercially available starting materials, compounds known in the literature, or readily prepared intermediates, by employing standard synthetic methods (in addition to those provided herein). Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be readily obtained from the relevant scientific literature or from standard textbooks in the field. It will be appreciated that where typical or preferred process conditions (e.g., reaction temperatures, times, mole ratios of reactants, solvents, pressures) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary7 with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures. Those skilled in the art of organic synthesis will recognize that the nature and order of the synthetic steps presented may be varied for the purpose of optimizing the formation of the compounds described herein.

[0093] Synthetic chemistry transformations (including protecting group methodologies) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R.C. Larock, Comprehensive Organic Transformations, 2d. Ed., Wiley-VCH Publishers (1999); P.G.M. Wuts and T.W. Greene, Protective Groups in Organic Synthesis, 4th Ed., John Wiley and Sons (2007); L. Fieser and M. Fieser, Fieser andFieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof, each of which are hereby incorporated by reference in their entirety.

[0094] The processes described herein can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 'H or 13C), infrared spectroscopy (FT-IR), spectrophotometry (e.g., UV-visible), or mass spectrometry (MS), or by chromatography such as high-pressure liquid chromatography (HPLC) or thin layer chromatography (TLC).

[0095] Preparation of compounds can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated herein by reference in its entirety.

[0096] The reactions of the processes described herein can be carried out in suitable solvents which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, i.e., temperatures which can range from the solvent’s freezing temperature to the solvent’s boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected.

[0097] Resolution of racemic mixtures of compounds can be carried out by any of numerous methods known in the art. For example, the absolute configuration of the stereoisomers may be determined by ID and 2D NMR techniques such as COSY, NOESY, HMBC and HSQC. Specific implementations of these NMR techniques may be found in Hauptmann, H et al., Bioconjugate Chern. 11 (2000): 239-252 or Bowler, J. Steroids 54 / 1 (1989): 71-99, each hereby incorporated by reference in their entirety. Another example method includes preparation of the Mosher’s ester or amide derivative of the corresponding alcohol or amine, respectively. The absolute configuration of the ester or amide is then determined by proton and / or 19F NMR spectroscopy. An example method includes fractional recrystallization using a “chiral resolving acid” which is an optically active, salt-forming organic acid. Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids, such as the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, or the various optically active camphorsulfonic acids. Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent compositions can be determined by one skilled in the art.

[0098] Oligonucleotides of the present disclosure (e.g., probe oligonucleotide, amplifier oligonucleotide, pre-amplifer oligonucleotide, primer oligonucleotide) may be prepared by any of a variety of methods (see, e.g., Sambrook et al., “Molecular Cloning: A Laboratory ManuaC, 1989, 2.sup.nd Ed., Cold Spring Harbour Laboratory Press: New York, N.Y.; “PCR Protocols: A Guide to Methods and Applications”, 1990, Innis (Ed.), Academic Press: New York, N.Y.; Tijssen “Hybridization with Nucleic Acid Probes—Laboratory Techniques in Biochemistry and Molecular Biology (Parts I and IL)”, 1993, Elsevier Science; “PCR Strategies”, 1995, Innis (Ed.), Academic Press: New York. N.Y.; and “Short Protocols in Molecular Biology”. 2002. Ausubel (Ed.). 5.sup.th Ed.. John Wiley & Sons: Secaucus, N.J.). each of which are hereby incorporated by reference in their entirety7.

[0099] In some embodiments, oligonucleotides may be prepared by chemical techniques including, e.g.. chemical synthesis and polymerization based on a template as described, e.g.. in Narang et al., Meth. Enzymol. 68:90-98 (1979); Brown et a)., Meth. Enzymol. 68: 109-151 (1979); Belousov et al., Nucleic Acids Res. 25:3440-3444 (1997); Guschin et al., Anal. Biochem. 250:203-211 (1997); Blommers et al., Biochemistry 33:7886-7896 (1994); Frenkel et al.. Free Radic. Biol. Med. 19:373-380 (1995); and U.S. Pat. No. 4,458,066. each of which are hereby incorporated by reference in their entirety.

[0100] In some embodiments, oligonucleotides may be prepared (including conjugation to the chemiluminescent moiety) using an automated, solid-phase procedure based on the phosphorami di te approach. In such methods, each nucleotide is individually added to the 5'- end of the growing oligonucleotide chain, which is attached at the 3'-end to a solid support. The added nucleotides are in the form of trivalent 3'-phosphoramidites that are protected from polymerization by a dimethoxytriyl (or DMT) group at the 5'-position. After base-induced phosphorami di te coupling, mild oxidation to give a pentavalent phosphotriester intermediate and DMT removal provides anew site for oligonucleotide elongation. The oligonucleotides are then cleaved off the solid support, and the phosphodiester and exocyclic amino groups are deprotected with ammonium hydroxide. These syntheses may be performed on oligo synthesizers such as those commercially available from Perkin Elmer / Applied Biosystems, Inc. (Foster City, Calif.), DuPont (Wilmington, Del.) or Milligen (Bedford, Mass.). Alternatively, oligonucleotides can be custom made and ordered from a variety of commercial sources well-known in the art, including, for example, the Midland Certified Reagent Company (Midland, Tex.), ExpressGen. Inc. (Chicago, Ill.), Operon Technologies, Inc. (Huntsville, Ala.), and many others.

[0101] Purification of oligonucleotides, where necessary or desirable, may be carried out by any of a variety of methods well-known in the art. For example, purification of oligonucleotides is typically performed either by native acrylamide gel electrophoresis, by anion-exchange HPLC, e.g., see Pearson and Regnier, J. Chrom. 255:137-149 (1983) or by reverse phase HPLC, e.g., see McFarland and Borer, Nucleic Acids Res. 7:1067-1080 (1979).

[0102] The sequence of oligonucleotides can be verified using any suitable sequencing method including, but not limited to, chemical degradation, e.g., see Maxam and Gilbert, Methods of Enzymology, 65:499-560 (1980), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, e.g., see Pieles et al., Nucleic Acids Res. 21:3191-3196 (1993). mass spectrometry following a combination of alkaline phosphatase and exonuclease digestions, e.g., see Wu and Aboleneen, Anal. Biochem. 290:347-352 (2001).

[0103] The present disclosure encompasses modified versions of these oligonucleotides that may perform as equivalents of these oligonucleotides in accordance with the methods of the present disclosure. These modified oligonucleotides may be prepared using any of several means known in the art. Non-limiting examples of such modifications include methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, and intemucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.), or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Modified oligonucleotide may also be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. The oligonucleotides of the present disclosure may also be modified with a label and, particularly, the probe oligonucleotide.

[0104] In some embodiments, the oligonucleotide (e.g., probe oligonucleotdie, primer) are labeled with a chemiluminescent detectable agent or moiety before being used in assays. The role of a detectable agent is to allow visualization and detection of amplified target sequences. Preferably, the detectable agent is selected such that it generates a signal which can be measured and whose intensity is related (e.g., proportional) to the amount of amplification products in the sample being analyzed.

[0105] The association between the oligonucleotide and the detectable agent can be covalent or non-covalent. Labeled detection primers can be prepared by incorporation of or conjugation to a detectable moiety. Labels can be attached directly to the nucleic acid sequence or indirectly (e.g.. through a linker). Linkers or spacer arms of various lengths are commercially available, and can be selected to reduce steric hindrance, or to confer other useful or desired properties to the resulting labeled molecules, e.g., see Mansfield et al., Mol. Cell Probes 9:145-156 (1995), which is hereby incorporated by reference in their entirety.

[0106] Various methods for labeling nucleic acid molecules are embraced by the present disclosure. For a review of exemplary labeling protocols, and label detection techniques, see, for example, Kricka, Ann. Clin. Biochem. 39:114-129 (2002); van Gijlswijk et al., Expert Rev. Mol. Diagn. 1:81-91 (2001); and Joos et al.. J. Biotechnol. 35:135-153 (1994), each of which are hereby incorporated by reference in their entirety. Standard nucleic acid labeling methods include: incorporation of radioactive agents, direct attachments of fluorescent dyes (Smith et d.,Nucl. Acids Res. 13:2399-2412 (1985)) or of enzymes (Connoly and Rider, Nucl. Acids. Res. 13:4485-4502 (1985)); chemical modifications of nucleic acid molecules making them detectable immunochemically or by other affinity reactions, e.g., see Broker et al., Nucl. Acids Res. 5:363-384 (1978); Bayer et al., Methods of Biochem. Analysis 26:1-45 (1980); Langer et al., Proc. Natl. Acad. Sci. USA 78:6633-6637 (1981); Richardson et al., Nucl. Acids Res. 11:6167-6184 (1983); Brigati et al., Virol. 126:32-50 (1983); Tchen et al„ Proc. Natl. Acad. Sci. USA 81:3466-3470 (1984); Landegent et al., Exp. Cell Res. 15:61-72 (1984); and Hopman et al., Exp. Cell Res. 169:357-368 (1987); and enzyme-mediated labeling methods, such as random priming, nick translation, PCR and tailing with terminal transferase. For a review on enzymatic labeling, see, e.g., Temsamani and Agrawal, Mol. Biotechnol. 5:223-232 (1996) , each of which are hereby incorporated by reference in their entirety. More recently developed nucleic acid labeling systems include, but are not limited to: ULS (Universal Linkage System), which is based on the reaction of monoreactive cisplatin derivatives with the N7 position of guanine moieties in DNA (Heetebrij et al., Cytogenet. Cell. Genet. 87:47-52 (1999)), psoralen-biotin, which intercalates into nucleic acids and upon UV irradiation becomes covalently bonded to the nucleotide bases (Levenson et al.. Methods Enzymol. 184:577-583 (1990); and Pfannschmidt et ai.,Nucleic Acids Res. 24:1702-1709 (1996)), photoreactive azido derivatives (Neves et al., Bioconjugate Chem. 11:51-55 (2000)), and DNA alkylating agents (Sebestyen et al., Nat. Biotechnol. 16: 568-576 (1998)) , each of which are hereby incorporated by reference in their entirety.

[0107] Typically, zwitterionic acridinium esters (“ZAE”) comprising a reactive functional group for forming covalent linkages as described in U.S. Pat Nos. 6,664,043 to Natrajan et al., 7,309,615 to Natrajan etal., 9,575.062 to Natrajan etal., or 9,487.480 to Natrajan, each hereby incorporated by reference in their entirety and in particular with respect to the zwitterionic acridinium esters described therein and their syntheses, may be used for synthesizing the compounds disclosed herein. For example, the zwitterionic acridinium ester starting materials may comprise an N-sulfopropyl (“NSP”) group in a zwitterionic moiety and / or comprise a charged nitrogen atom connected to the charged acridinium nucleus (“DIZAE”) and / or comprise a sterically stabilized dimethyl acridinium ester (“DMAE”) and / or comprise an isopropoxy functionalized aciridinium nucleus (“ISO”) and / or comprise a zwitterionic (“Z”) and / or hexa(ethylene) glycol derived (“HEG”) and / or glutarate derived (e.g., -C(O)-(CH2)3-C(O)-) linking moieties between the acridinium ester and the reactive functional group. The reactive functional group may by NH2 or N-hydroxysuccinimidyl ester (“NHS”). For example, the compound (e.g., a compound for conjugating with an analyte or binding partner of an analyte such as a peptide, a protein, or a macromolecule including an antibody) may have the structure of formula (I): RFG--L--T wherein RFG is a reactive functional group for conjugating to the analyte or binding partner for an analyte, L is absent (i.e., it is a bond) or a linker, and T is a chemiluminescent acridinium. The chemiluminescent conjugates or compounds for forming the conjugates may also be synthesized through the use of acridinium sulfonamide reactants. For example, the acridinium sulfonamides disclosed in US Pat No 5,543,524 to Mattingly et al., hereby incorporated by reference in its entirety, are useful starting materials for the preparation of the chemiluminescent compounds disclosed herein.

[0108] The particle in the solid phase, and how it is prepared during the course of the assay may be important to achieve the desired resolution of the assay. The particles may have an average diameter of at least about 0.02 microns and not more than about 100 microns. In some embodiments, the particles have an average diameter from about 0.05 microns to about 20 microns, or from about 0.3 microns to about 10 microns. The particle may be organic or inorganic, swellable or non-swellable, and porous or non-porous. In a particular (but not limiting) embodiment, the particle has a density approximating water, generally from about 0.7 g / mL to about 1.5 g / mL. and is composed of material that can be transparent, partially transparent, or opaque. The particles can be comprised of organic and inorganic polymers, latex particles, magnetic or non-magnetic particles, and the like. In some non-limiting examples, the particles are chrome particles or latex particles.

[0109] The polymer particles can be formed of addition or condensation polymers. The particles can also be derived from naturally occurring materials, naturally occurring materials that are synthetically modified, and synthetic materials. Among organic polymers of particular interest are polysaccharides, particularly cross-linked polysaccharides, such as (but not limited to) agarose, which is available as Sepharose; dextran, which is available as Sephadex and Sephacryl; cellulose; starch; and the like; addition polymers, such as polystyrene, polyvinyl alcohol, homopolymers and copolymers of derivatives of acrylate and methacrylate, particularly (but not limited to) esters and amides having free hydroxyl functionalities, and the like.

[0110] The particles are typically readily dispersible in an aqueous medium and can be adsorptive or functionalizable so as to permit conjugation to monoclonal antibodies (or fragments thereof), either directly or indirectly through a linking group. When a linking group is utilized, in some non-limiting embodiments, any linking group may comprise about 2 to about 50 atoms, or 4 to about 30 atoms, not counting hydrogen and may comprise a chain of from 2 to about 30 atoms, or 3 to about 20 atoms, each independently selected from the group normally consisting of carbon, oxygen, sulfur, nitrogen, and phosphorous. In some examples, the linking group comprises an oxime functionality. [OlH] The number of heteroatoms in the linking group may be in the range from 0 to about 20, or 1 to about 15, or about 2 to about 10. The linking group may be aliphatic or aromatic. When heteroatoms are present, oxygen is normally present as oxo or oxy. bonded to carbon, sulfur, nitrogen or phosphorous, nitrogen is normally present as nitro, nitroso or amino, normally bonded to carbon, oxygen, sulfur or phosphorous; sulfur is analogous to oxygen; while phosphorous is bonded to carbon, sulfur, oxygen or nitrogen, usually as phosphonate and phosphate mono- or diester. Common functionalities in forming a covalent bond between the linking group and the molecule to be conjugated are alkylamine, amidine, thioamide, ether, urea, thiourea, guanidine, azo, thioether and carboxylate, sulfonate, and phosphate esters, amides and thioesters.

[0112] For the most part, when a linking group has a linking functionality (functionality for reaction with a moiety which includes cross-linking functionality) such as, for example, anon-oxocarbonyl group including nitrogen and sulfur analogs, a phosphate group, an amino group, alky lating agent such as halo or tosylalkyl, oxy (hydroxyl or the sulfur analog, mercapto), oxocarbonyl (e.g., aldehyde or ketone), or active olefin such as a vinyl sulfone or a-, (3unsaturated ester, these functionalities may be linked to amine groups, carboxyl groups, active olefins, alkydating agents, e.g., bromoacetyl. Where an amine and carboxylic acid or its nitrogen derivative or phosphoric acid are linked, amides, amidines, and phosphoramides may be formed. Where mercaptan and activated olefm are linked, thioethers are formed. Where a mercaptan and an alkylating agent are linked, thioethers may be formed. Where aldehyde and an amine are linked under reducing conditions, an alkylamine may be formed. Where a ketone or aldehyde and a hydroxylamine (including derivatives thereof where a substituent is in place of the hydrogen of the hydroxyl group) are linked, an oxime functionality (=N—O—) may be formed. Where a carboxylic acid or phosphate acid and an alcohol are linked, esters may be formed. Various linking groups are provided in, for example, Cautrecasas, J. Biol. Chern. (1970): 245:3059, which is hereby incorporated by reference in its entirety7 and particularly in relation to linking groups.

[0113] A kit according to the present disclosure typically involves an immunoassay reagent composition comprising an AE conjugated to a probe oligomer. The kit may comprise an immunoassay reagent composition comprising a preamplifier oligomer. The kit may comprise an immunoassay reagent composition comprising an amplifier oligomer. In various implementations, particularly of the Lite Reagents describved herein, the kit may comprise an immunoassay reagent composition comprising a label probe oligomer conjubated to an acridinium ester and an amplifier oligomer. In various implementations, the kit may comrpise one or more reagenent compositions comprising The kit may comprise accessory ingredients such a buffers, blocking reagents, ions, e.g. bivalent cations or monovalent cations, calibration proteins, secondary' antibodies, detection reagent such as detection dyes and any other suitable compound or liquid necessary for the performance of analyte detection. Additionally, the kit may comprise an instruction leaflet and / or may provide information as to the relevance of the obtained results. In some embodiments, the kit further comprises the solid phase reagent. In some embodiments, the kit further comprises chemiluminescence triggering reagents.

[0114] A variety of immunoassay formats, including, for example, competitive and noncompetitive immunoassay formats, antigen / analyte capture assays, and two-antibody sandwich assays can be used in accordance with the cartridges, kits, and methods described herein. The assay may be, for example, a competitive immunoassay which typically involves the detection of a large molecule, also referred to as macromolecular analyte, using binding molecules such as antibodies. The antibody is immobilized or attached to a solid phase such as a particle, bead, membrane, microtiter plate, or any other solid surface.

[0115] In an example of a competitive heterogeneous assay, a support having an antibody for an analyte (e.g., bovine monoclonal antibodies, mouse monoclonal antibodies, antibody fragments such as bovine antibody fragments, mouse antibody fragments) bound thereto is contacted with a medium containing a sample suspected of containing the analyte and the chemiluminescent conjugates (or “labeled analogs”) described herein. Analyte from the sample may compete for binding to the analyte antibody with the labeled analog. After separating the support and the medium, the label activity of the support or the medium is determined by conventional techniques and is related to the amount of analyte in the sample. In a variation of the above competitive heterogeneous assay, the support comprises the analyte analog, which competes with analyte of the sample for binding to an antibody reagent in accordance with the principles described herein. The labeled analyte analog may be covalently attached with a chemiluminescent or fluorescent molecule often referred to as a label or tracer.

[0116] When the solid phase with the immobilized antibody is mixed with a sample containing the analyte and the labeled analyte, a binding complex is typically formed between the analyte or the labeled analyte. This type of assay is often called a heterogeneous assay because of the involvement of a solid phase. The chemiluminescent signal associated with the binding complex can then be measured and the presence or absence of the analyte in the sample can be inferred. Usually, the binding complex is separated from the rest of the binding reaction components such as excess, labeled analyte, prior to signal generation. For example, if the binding complex is associated with a magnetic bead, a magnet can be used to separate the binding complex associated with the bead from bulk solution.

[0117] In an example of a sandwich assay format employing two antibodies (or fragments thereof), a solid support with a first immobilized antibody or fragment thereof for an analyte is mixed with a sample containing the analyte and a labelled conjugate comprising a second antibody or fragment thereof. A binding complex is formed between the solid particle and the labelled conjugate via the analyte in the sample. The signal associated with the binding complex and can the measured and the presence or absence or amount of analyte can be inferred. Usually, the binding complex is separated from the rest of the binding reaction components such as excess, labeled analyte, prior to signal generation. For example, if the binding complex is associated with a magnetic bead, a magnet can be used to separate the binding complex associated with the bead from bulk solution. In some embodiments, the first immobilized antibody is a biotinylated mouse monoclonal antibody bound to coated (e.g., streptavidin coated) optionally paramagnetic particles. In some embodiments, the second antibody is a mouse monoclonal antibody fragment labelled with acridinium (e.g., acridinium ester). FIG. 1A provides a schematic of an exemplary acridinium ester based sandwich immunoassay illustrating the conjugation of a primary antibody to both the magnetic particle and target (e.g., analyte), and the conjugation of the secondary antibody to another portion of the target and an acridinium ester. FIG. IB is a schematic of a branched DNA configuration of this sandwich assay. As can be seen, a biological sample (comprising the target molecule) is added to a mixture of the magnetic particle (M) having the primary antibody (or the “catcher antibody'’) conjugated thereto and a secondary antibody having the preamplifer oligo conjugated thereto (or the “detection antibody-preamplifer conjugate”). The target molecule becomes conjugated to both antibodies and bound to the magnetic particle. The b-DNA alimplifer oligo is added to the mixture affording conjugation of multiple b-DNA amplifier oligos to the detection antibody-preamplifier conjugate. Subsequently, addition of the DNA label probe oligos conjugated to a chemiluminescent moiety (such as a chemiluminescent acridinium) results hybridization between the amplifier oliogs and the probe oligos resulting in the presence of multiple chemiluminescent moietes per analyte bound to the magnetic particle.

[0118] By using a series of‘‘standards,” that is, known concentrations of the analyte, a “doseresponse” curve can be generated for the known labeled analyte. These dose response curves may be identified individually for any acridinium label or identified based on combinations of the acridinium labels used in the assay. Thus, the dose-response curve correlates a certain amount of measured signal with a specific concentration of analyte. In a competitive assay, as the concentration of the analyte increases, the amount of signal decreases if the chemiluminescence from the binding complex is measured. The concentration of the analyte in an unknown sample can then be calculated by comparing the signal generated by an unknown sample containing the macromolecular analyte, with the dose-response curve.

[0119] The methodology of the attachment of binding molecules such as antibodies to solid phases ty pically involves a mixing of the requisite components to induce attachment. For example, an antibody can be covalently attached to a particle containing amines on its surface by using a cross-linking molecule such as glutaraldehyde. The attachment may also be non-covalent and may involve simple adsorption of the binding molecule to the surface of the solid phase, such as polysty rene beads and microtiter plate. Labeling of binding molecules such as antibodies and other binding proteins may be referred to as conjugation reactions and the labeled antibody is often called a conjugate. Typically, an amine-reactive moiety on the label reacts with an amine on the antibody to form an amide linkage. Other linkages, such as thioether, ester, carbamate, and the like between the antibody and the label may also be used.

[0120] In another aspect of the invention, a reagent may be provided for the detection of an analyte comprising a chemiluminescent acridinium compound bound to the probe-oliogo (which may be referred to herein as a Lite Reagent). The reagent may comprise from 0.1 to 100 ng / mL of the chemiluminescent acridinium compound conjugated to a probe oliogomer or from 1 to 50 ng / mL of the chemiluminescent acridinium compound to a probe oliogomer or from 5 to 30 ng / mL of the chemiluminescent acridinium compound to a probe oliogomer. In some embodiments, the compound is provided in a reagent which further comprises a buffer. In some embodiments, the reagent (e.g., Lite Reagent) comprises an amplifier oligomer. In various implementations, a reagent may comprise a preamplifier oligomer. In certain implementations, the reagent (e.g., Solid Phase Reagent) comprises a particle (e.g., straptavidin-coated magnetic beads) having an antibody or antibody fragment that binds to the analyte attached to the surface of the particle.

[0121] In some embodiments, the sample derived from a mammal (e.g., human). In some embodiments, the sample comprises saliva and / or blood and / or serum. In some embodiments, the sample is saliva and / or blood and / or serum.

[0122] In some assays, the sample to be analyzed is subjected to a pretreatment to release analyte from endogenous binding substances such as, for example, plasma or serum proteins that bind the analyte. The release of the analyte from endogenous binding substances may be carried out. for example, by addition of a digestion agent or a releasing agent or a combination of a digestion agent and a releasing agent used sequentially. The digestion agent is one that breaks down the endogenous binding substances so that they can no longer bind the analyte.

[0123] The conditions for conducting an assay on a portion of a sample in accordance with the principles described herein may include carrying out the assay in an aqueous buffered medium at a moderate pH, generally that which provides optimum assay sensitivity. The aqueous medium may be solely water or may include from 0.1 to 40 % by volume of a cosolvent. The pH for the medium may be in the range of 4 to 11, or 5 to 10, or 6.5 to 9.5, or 7 to 8. Usually, the pH value of the solution will be a compromise between optimum binding of the binding members of any specific binding pairs, the pH optimum for other reagents of the assay such as members of the signal producing system, and so forth. Various buffers may be used to achieve the desired pH and maintain the pH during the assay. Illustrative buffers include borate, phosphate, carbonate, TRIS, barbital, PIPES, HEPES, MES, ACES, MOPS, and BICINE, for example.

[0124] Various ancillary' materials may be employed in the assay methods. For example, in addition to buffers, the composition, reagents, or reaction medium may comprise stabilizers for the medium and for the reagents employed. In some embodiments, the medium may comprise proteins (e.g., albumins), organic solvents (e.g., formamide), quaternary ammonium salts, polyanions (e.g., dextran sulfate), binding enhancers (e.g., polyalkylene glycols), polysaccharides (e.g., dextran, trehalose), blockers (e.g., blocker antibodies to prevent false positives), and combinations thereof.

[0125] Triggering the chemiluminescence of the analogs may be performed by the addition chemiluminescent triggering reagents. The chemiluminescent triggering reagents may be acidic or basic. Multiple chemiluminescent triggering reagents may be added sequentially. For example, an acidic solution (e.g.. Acid Reagent) may first be added followed by a basic solution (e.g., Basic Reagent). In some embodiments, the chemiluminescent triggering reagents comprise hydrogen peroxide, hydrogen peroxide salts, nitric acid, nitric acid salts, sodium hydroxide, ammonium salts, or combinations thereof. EXAMPLES

[0126] The following Examples illustrate the synthesis of a representative number of compounds, characterization of parameters implicated in assay development, and the use of these compounds in the measurement of samples in heterogeneous competitive assay. Accordingly, the Examples are intended to illustrate but not to limit the disclosure. Additional compounds not specifically exemplified may be synthesized using conventional methods in combination with the methods described herein.

[0127] Example 1: Comparison of Assay Formats

[0128] The Limit of Detection (LOD) for various targets using the standard sandwich immunoassay format such as that described in FIG. 1 A, a single molecule array (SiMOA) such as that available from Quanterix, and the branched DNA assays of the present disclosure, such as that depicted in FIG. IB, was assessed. Table 1 provides the LOD for each assay illustrating the advantages offered by branched DNA assays to standard sandwich immunoassays. Moreover, branched DNA sandwich immunoassays provide limits of detection comparable to single molecule assays and PEA assay without the problems associated therewith. Table 1 Target Sandwich Immunoassay (pg / mL) Expected via b-DNA (pg / mL) SiMoA (pg / mL) IL-6 3.0 0.06-0.15 0.01 Troponin I 2.5 0.05-0.13 0.079 NFL 2.5 0.05-0.13 0.174

[0129] Example 2: Alkaline Phosphatase Branched DNA Assay

[0130] Using alkaline phosphatase based label probes, three different assay formats were assesed. FIG. 2 provides schematics of these formats involving conjugation using an alkaline phosphatase probe only (1), conjugation of an amplifier to several alkaline phosphatase probes (2). and conjugation with branched DNA using a preamplifier, amplifier and alkaline phosphatase probe (3).

[0131] Table 2 provides the signal, noise, and signal to noise ratio (S / N) for each assay format provided in RLUs. As can be seen, the signal increases in assay formats involving the amplifier (2, 3) and the preamplifer (3) are observed without an increase in the noise. Without wishing to be bound by theory, this results in higher signal to noise ratios for the branched DNA assays. Table 2 Assay Format Signal Noise S / N 1. Alkaline Phosphatase Probe 2.6 0.4 5.5 2. Amplifier + Alkaline Phosphatase Probe 10.3 0.5 19.6 3. Preamplifier + Amplifier + Alkaline Phosphatase Probe 93.2 0.6 154.3

[0132] Example 3: Proof of Concept Signal Amplification Assay

[0133] A three-component hybridization assay using direct chemiluminometric technology and branched DNA signal amplication was developed and performed. A schemtaic description of the assay is shown in FIG. 3. As can be seen, the the following steps were performed: dispensing of 100 pL sample (PreAmplifier Reagent) into cuvette; dispensing of 100 pL Lite Reagent (Amplifier, Label Probe) into cuvette and incubates for 12 minutes at 37°C dispensing of 50 pL Solid Phase Reagent (Streptavidin-coated magnetic beads) into cuvette and incubates for 5 minutes at 37°C separation, aspiration, and washing of the cuvettes with Wash buffer dispensing 300 pL each of Acid Reagent and Base Reagent to trigger the chemiluminescent reaction. These experiments validated three-component hybridization. In these experiments, the preamplifer reagent and lite reagent contained sodium citrate, pH 7, sodium chloride, bovine serum alubmin (fraction V), polyvinylpyrrolidone K-30, Ficoll 400, Formamide, Blocking reagent (Roche). The solid phase reagent contained a sodium phosphate buffer, pH 7.2, sodium chloride, and candor blocking solution.

[0134] The following oligomers were used: PreAmplifier (PreAmp) 5‘ -TTT GGA AAG AAA GTG AAG TGT AGG CAT AGG ACC CGT GTC TTT TTT AGG CAT AGG ACC CGT GTC CGT GGA TGT TTG AGG CAT AGG ACC CGT GTC TTT TTT-Biotin-3‘ (SEQ ID NO 15) Amplifier (Amp) 5‘ -TGA CAC GGG TCC TAT GCC TTT TAG TCA GCG CCG TAC CAA GTG CTT TTT TAG TCA GCG CCG TAC CAA GTG CTT TTT TAG TCA GCG CCG TAC CAA GTG CTT TTT T-3‘ (SEQ ID NO 12) Label Probe (LP) 5-GCA CTT GGT ACG GCG CTG ACT TTT-AE-3‘ (SEQ ID NO 16) where AE indicates acridinium ester conjugation (e.g., DMAE). The solidphase particle was Dynabeads™ My One™ Streptavidin Cl and analysis was performed on an Atellica® Immunoassay Analyzer IM 1600 with an Atellica® Sample Handler. Additionally, for comparison a probe label (PL) was used having the sequence 5-ACA CTT CAC TTT CTT TCC AA-AE-3‘ (SEQ ID NO 17) where AE is the same acridinium as present in the label probe. In the sequences above, complementary' sequences are identified with the simlar underline style (i.e., complementary sequences in the preamplifier and probe label are dash underlined, complementary sequences in the preamplifier and the amplifier are single underlined, and complementary sequences in the amplifier and label probe are double underlined).

[0135] The AE had the structure (as linked to the 5’ ends of these oligomers) and was conjugated to each oligomer in the following manner: 5'-end vTVWVW where B was the 3’ base of the oligomer (A in the probe label, T in the label probe).

[0136] To compare the signal amplification afforded with the branched DNA as described in the present application, 3 different Lite reagents were developed: two component systems (“2com”), three component systems “3 com / ’ and three component systems with the amplifier oligomer (3 com-Amp). The 2com Lite reagent contained 10 nM probe label (PL) oligomer which performs a two component hybridization with the preamplifier in the sample (dashed underlined). The 3com Lite reagent contains 4 nm Amplifier oligomer and 10 nM label probe (LP) oligomer, which performs a 3-components hybridization with preamplifier. In the hybridication and signal amplificaiton of the 3com Lite reagent, the label probe has a total of nine binding sites on the bDNA complex. The 3com-Amp Lite reagent is the 3com Lite reagent without the the amplifier (i.e., 10 nM LP without amplifier) and therefore cannot bind to the preamplifier.

[0137] The measured chemiluminescence (in relative light untis (RLU)) from each of these assay formats is provided in FIGS. 4A and 4B. These figures show the average RLU measured, as a function of preamplifier concentration, after two repeated measurements (error bars represent the standard deviation). Due to the assay format, the preamplifier oligo concentration is a proxy for analyte concentration. As can be seen, signal amplification occurs when using the assay formats of the present disclousre. In this case, chemiluminescent signal is increased by 90% in the low measurement range (under 1 pM) and signal is increased overall by 80%.

[0138] Increasing the branching and binding sites increases the chemiluminescent signal. For example, in an HIV-1 RNA assay, 196 binding sites can be generated with this technology which is expected to lead to more than 20* enhancement of chemiluminescent signal. NON-LIMITING ILLUSTRATIVE EMBODIMENTS

[0139] Non-limiting illustrative embodiments are provided below, each of which should be considered to be part of the disclosure of the present application. These embodiments may apply to any embodiment described herein.

[0140] Non-limiting illustrative embodiment 1. A method for the detection or quantification of an analyte in a sample (e.g., a biological sample such as blood, saliva, serum, a sample derived from a biological sample such as a diluted biological sample) comprising: (a) mixing the sample with a composition comprising a branched DNA (b-DNA) pre-amplifier oligomer conjugated to a first antibody or antibody fragment that binds to the analyte and optionally a particle having a second antibody or antibody fragment that binds to the analyte attached to the surface of the particle: (b) adding to the mixture a particle having a second antibody or antibody fragment that binds to the analyte attached to the surface of the particle (e.g., if the particle is not already present in the mixture); (c) adding to the mixture a b-DNA amplifier oligomer that binds to the preamplifier oligomer, wherein, optionally, multiple amplifier oligomers bind to at least one of the pre-amplifier oligomers conjugated to the analy te; (d) adding to the mixture a label probe comprising a chemiluminescent moiety bound to a probe oligomer, wherein the probe oligomer binds to one of the amplifier oligomers and optionally, multiple probe oligomers bind to the same amplifier oligomer; (e) preparing the mixture to measure chemiluminescence (e.g., by separation of the particle having chemiluminescent acridiniums conjugated to the surface from the mixture); (I) triggering chemiluminescence from the prepared mixture; and (g) detecting the presence or calculating the concentration of said at least one analyte by comparing the amount of chemiluminescence with a standard dose response curve which relates the amount of light emitted to a known concentration of the at least one of the multiple analytes.

[0141] Non-limiting illustrative embodiment 2. The method according to Non-limiting illustrative embodiment 1, the analyte is an exosome (e.g., neuron-derived, astrocyte-derived, oligodendrocyte-derived, microglia-derived) protein biomarker.

[0142] Non-limiting illustrative embodiment 3. The method according to Non-limiting illustrative embodiment 1 or 2, wherein the analyte is glypican-1 (GPC1), neurofilament light chain (NFL), phosphorylated tau (p-tau), or amyloid beta (A(3).

[0143] Non-limiting illustrative embodiment 4. The method according to any one ofNon-limiting illustrative embodiments 1-3, wherein the probe oligomer is a an oligonucleotide comprising the sequence 5 -AGT FAJ CGC FGT AFT T-3‘ (SEQ ID NO 9) or a sequence having more than 80% (e.g., more than 90%, more than 95%) identity therewith; wherein F is isocytosine and J is isoguanine.

[0144] Non-limiting illustrative embodiment 5. The method according to any one of Nonlimiting illustrative embodiments 1-4, wherein the amplifier oligomer is an oligonucleotide comprising the sequence 5'-TTT-(JTA CJG CGF TJA CT TTTTT)n-TCF ACG JCF CTA JG-3' (SEQ ID NO 10) or a sequence having more than 80% (e.g., more than 90%, more than 95%) identity therewith; wherein n is an integer (e.g., an integer from 1-50 or 1-20 or 2-50 or 2-20 or 2-15 or 5-15); F is isocytosine and J is isoguanine.

[0145] Non-limiting illustrative embodiment 6. The method according to any one of Nonlimiting illustrative embodiments 1 -5, wherein the pre-amplifier oligomer is an oligonucleotide having the sequence 5'-TTT-(CFT AGJ GFC GTJ GA TTTTT)n-3' (SEQ ID NO 11) or a sequence having more than 80% (e.g.. more than 90%, more than 95%) identity therewith; wherein n is an integer (e.g., an integer from 1-50 or 1-20 or 2-50 or 2-20 or 2-15 or 5-15); F is isocytosine and J is isoguanine.

[0146] Non-limiting illustrative embodiment 7. The method according to any one of Nonlimiting illustrative embodiments 1-6, wherein the sample is blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cervical swab, tears, saliva, buccal swab, skin, brain tissue, or cerebrospinal fluid, or derived therefrom (e.g., diluted sample).

[0147] Non-limiting illustrative embodiment 8. The method according to any one of Nonlimiting illustrative embodiments 1-7, wherein the first antibody or antibody fragment is a mouse monoclonal antibody or fragment thereof (e.g., F(ab)).

[0148] Non-limiting illustrative embodiment 9. The method according to any one ofNon-limiting illustrative embodiments 1-8, wherein the linker comprises (or is) polyethylene glycol (PEG).

[0149] Non-limiting illustrative embodiment 10. The method according to any one ofNon-limiting illustrative embodiments 1-9, wherein the label probe is added in molar excess of preamplifier oligomer (e.g., less than 100x excess or less than 50* excess or less than 40* excess or less than 30* excess or from 5* excess to 25x excess) and / or the label probe is added in molar excess (e.g., less than 100x excess or less than 50x excess or less than 40x excess or less than 30x excess or from 5 x excess to 25 x excess) of the amplifier oligomer and / or the amplifier oligomer is added in molar excess of the pre-amplifier oligomer (e.g.. less than 100x excess or less than 50x excess or less than 40x excess or less than 30x excess or from 5x excess to 25x excess).

[0150] Non-limiting illustrative embodiment 11. The method according to any one of Nonlimiting illustrative embodiments 1-10, wherein the chemiluminescent moiety is an acridium or an alkaline phosphatase.

[0151] Non-limiting illustrative embodiment 12. The method according to any one of Nonlimiting illustrative embodiments 1-11, wherein the label probe was formed by i) reacting a chemiluminescent acridinium compound comprising a reactive functional group with a linking compound (e.g., a compound comprising a linker with reactive functional groups on each end, a compound already conjugated to the probe oligomer) and ii) reacting the probe oligomer to the linking compound.

[0152] Non-limiting illustrative embodiment 13. The method according to Non-limiting illustrative embodiment 12, wherein the chemiluminescent acridinium compound comprising a reactive functional group has the structure: RFGLT (I) wherein RFG is a reactive functional group for conjugation to the probe oligomer, L is absent (i.e., it is a bond) or a linker optionally comprising a group Lc or ZL, and 'P is a chemiluminescent acridinium comprising the structure: and “Zf’ are independently 0 (e.g., all R2 groups are hydrogen, all R3 groups are hydrogen), 1, 2, 3, or 4; Ri is hydrogen, -R, -Xb, -RL-Xb, -Lc-R, -Lc-Xb (e.g, -Li-Xb), -Z, -RL-Z, -Lc-Z (e.g., -Li-Z), or-Rl-Lc-Rl-Z (e.g., -Rl-Li-Rl-Z); R2 and R3 are independently selected at each occurrence from hydrogen, -R, an electron donating group, -Xc, -RL-XC, -Lc-Xc (e.g., -Li-Xc), and -Z; wherein two vicinal R2 or R3 groups may together form a fused cyclic group (e.g., 5-7 membered fused aryl or heteroaryl group, 5-7 membered fused heterocyclic group) and wherein R2 or Rs may comprise a linkage to an imaging agent such as a fluorophore (e.g., rhodamine); Lc is a divalent C1-35 alky l, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted (e.g., with 1 to 20 heteroatoms, with 1-20 substituents); ZL is a zwitterionic linker group having the structure: Xa “m” is 0 (i.e. it is a bond) or 1; “o’" and "p"’ are independently at each occurrence an integer from 0 (i.e. it is a bond) to 10: Z is a zwitterionic group independently at each occurrence has the structure: “q' and “F are independently 0 or 1; “r” is independently an integer from 0 to 10 (e.g., from 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); Xa and Xb are independently at each occurrence an anionic group; Xc is a protonated anionic group; Li is independently at each occurrence -0-, -S-, -NH-, -N(Rn)-, -(CH2)i-io-, -S(=O)i-2--0=0-, -C=C-(CH2)i-3-, -0(0)-, -O-C(O)-, -C(O)-(CH2) 1-4-, -(CH2) 1-4-0(0)-, -0(0)-0, -C(0)-N(Rn)-, -C(0)-NH-, -N(Rn)-C(O)-, -NH-C(O)-, -C(O)-N(Rn)-(CH2)i-3-, -(CH2)i-3-C(O)-N(Rn)- -(CH2)i-3-N(Rn)-C(O)-, -NH-S(O)i-2-. -N(Rn)-S(0)1-2-, -S(O)i-2-N(Rn)-, -S(O)i-2-NH- -(CH2)i-3-NH-S(O)i-2-, -(CH2)i-3-N(Rn)-S(O)i-2-, -(CH2)i-3-S(O)i-2-N(Rn)- -(CH2)i-3-S(O)i-2-NH-, -O-(CH2)i-4- -(CH2)i-4-O-, -S-(CH2)i-4- -(CH2)i-4-S-, -NH-(CH2)i-4-, -N(Rn)-(CH2)i-4-, -(CH2)i-4-N(Rn)-, -(OCH2)i-io-, -(CH20)i-io-, -(OCH2CH2)i-io-, or -(CH2CH20)i-io-; Rl is independently at each occurrence a C1-20 bivalent hydrocarbon radical (e.g.. alkyl, alkenyl, aryl, phenyl, mono alkyd substituted phenyl, di alkyl substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g, 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms, with 1-10 substituents); R is independently at each occurrence hydrogen or C1-35 hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms, with 1-20 substituents); R’ and R’’ are independently at each occurrence hydrogen or a C1-10 alkyl; Rn is independently at each occurrence from hydrogen or C1-5 alkyl (e.g, methyl, ethyl, propyl); and R’ is hydrogen or a C1-10 alkyl; or a salt thereof (e.g, a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxylate salt).

[0153] Non-limiting illustrative embodiment 14. The method according to any one of Nonlimiting illustrative embodiments 12-13, wherein the chemiluminescent acridinium comprising a reactive functional group has the structure of formula (la): (la) wherein Q is O or N; Y is selected from -R or -RL-Z, or in the case where Q is O then Y is absent; and Y’ is either absent (i.e. it is a bond), or is selected from -Li-, -RL-, -Rl-Li-, -L1-L1-, Li RL , Li RL Li, and RL Li RL .

[0154] Non-limiting illustrative embodiment 15. The method according to any one of Nonlimiting illustrative embodiments 12-14, wherein the chemiluminescent acridinium comprising a reactive functional group has the structure of formula (la) or (lb): wherein R4-R7 are independently hydrogen, an electron donating group, or C1-35 alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, or amino; and Y” is either absent (i.e., it is abond) or-Lc-, -Li- -RL-, or-RL-Li-.

[0155] Non-limiting illustrative embodiment 16. The method according to any one of Nonlimiting illustrative embodiments 13-15, wherein L is a C1-C5 alkylene.

[0156] Non-limiting illustrative embodiment 17. The method according to any one of Nonlimiting illustrative embodiments 12-16, wherein the reactive functional group of said chemiluminescent acridinium comprising a reactive functional group is an N-succinimidyl ester.

[0157] Non-limiting illustrative embodiment 18. The method according to any one of Nonlimiting illustrative embodiments 13-17. wherein R2 and R3 are independently -Xc, -RL-XC, -Lc-Xc (e.g, -Li-Xc).

[0158] Non-limiting illustrative embodiment 19. The method according to any one of Nonlimiting illustrative embodiments 13-17, wherein R2 and R3 are independently alkoxy (e.g., Ci-C4 alkoxy) substituted with -C(O)OH. -SO2OH). -OSO2OH), -OP(O)(ORp)OH, -OH, or combinations thereof.

[0159] Non-limiting illustrative embodiment 20. The method according to any one ofNon-limiting illustrative embodiments 1-19, wherein the second antibody or antibody fragment is a biotinylated antibody or antibody fragment.

[0160] Non-limiting illustrative embodiment 21. The method according to any one of Nonlimiting illustrative embodiments 1-20, wherein the particle is streptavidin coated.

[0161] Non-limiting illustrative embodiment 22. The method according to any one of Nonlimiting illustrative embodiments 1-21, wherein said detecting step can detect a difference in concentration of less than (or from 0.005 pg / mL to) 5 pg / mL (e.g., less than 1 pg / mL. less than 0.5 pg / mL. less than 0.2 pg / mL, from 0.01-0.2 pg / mL).

[0162] Non-limiting illustrative embodiment 23. The method according to any one ofNon-limiting illustrative embodiments 1-22, wherein said preparing step comprises separating the particle from the mixture and the chemiluminescence is triggered from the particle or the separated mixture.

[0163] Non-limiting illustrative embodiment 24. The method according to any one of Nonlimiting illustrative embodiments 1-23, wherein the method further comprising a washing step between the mixing step and any of the adding steps.

[0164] Non-limiting illustrative embodiment 25. The method according to any one of Nonlimiting illustrative embodimetns 1 -24, wherein the preamplifier oligomer may have the structure: 5 -TS-OL-(OL-C S ‘-OL^-OL-TS-S ’ the amplifier oligomer may have the structure: S-TS-OL-CS^-OMOL-CSVOL-TS-S’ the probe oligomer may have the structure: 5'-TS-CS2*-TS-3' where m and n are independently from 1-20 (e.g., 2-10, 2-5) TS is independently at each occurence absent or a sequence of nucleic acid residues (e.g., of from 1-15 bases in length) and may cap the indicated oligomer (e.g., preamplifier oligomer, amplifier oligomer, probe oligomer); OL is independently at each occurrence absent or a sequence of nucleic acid residues (e.g., of from 1-10 bases in length); CS1 is a complementary sequence of CS1*; and CS2 is a complementary sequence of CS2*.

[0165] Non-limiting illustrative embodiment 26. The method according to Non-limiting illustrative embodiment 25, wherein CS1 is selected from: AGT FAJ CGC FGT AF (SEQ ID NO 1) TCA JTF GCG JCA TJ (SEQ ID NO 2) CFT AGJ GFC GTJ GA (SEQ ID NO 3) GJA TCF CJG CAF CT (SEQ ID NO 4) AGG CAT AGG ACC CGT GTC (SEQ ID NO 5) TCC GTA TCC TGG GCA CAG (SEQ ID NO 6) GCA CTT GGT ACG GCG CTG ACT (SEQ ID NO 7), or CGT GAA CCA TGC CGC GAC TGA (SEQ ID NO 8).

[0166] Non-limiting illustrative embodiment 27. The method according to any one of Nonlimiting illustrative embodiments 25 or 26, wherein CS is selected from: AGT FAJ CGC FGT AF (SEQ ID NO 1) TCA JTF GCG JCA TJ (SEQ ID NO 2) CFT AGJ GFC GTJ GA (SEQ ID NO 3) GJA TCF CJG CAF CT (SEQ ID NO 4) AGG CAT AGG ACC CGT GTC (SEQ ID NO 5) TCC GTA TCC TGG GCA CAG (SEQ ID NO 6) GCA CTT GGT ACG GCG CTG ACT (SEQ ID NO 7), or CGT GAA CCA TGC CGC GAC TGA (SEQ ID NO 8).

[0167] Non-limiting illustrative embodiment 28. An immunoassay composition comprising a chemiluminescent label conjugated to a probe oligomer; and a earner or excipient.

[0168] Non-limiting illustrative embodiment 29. The immunoassay composition according to Non-limiting illustrative embodiment 28, further comprising an amplifier oligomer.

[0169] Non-limiting illustrative embodiment 30. The immunoassay composition according to Non-limiting illustrative embodiment 29, wherein the the amplifier oligomer has the structure: S’-TS-OL-CS^-OL-tOL-CSVOL-TS-S’, and the probe oligomer may have the structure: 5’-TS-CS2*-TS-3’ where n is from 1-20 (e.g., 2-10, 2-5), TS is independently at each occurence absent or a sequence of nucleic acid residues (e.g., of from 1-15 bases in length) and may cap the indicated oligomer (e.g., preamplifier oligomer, amplifier oligomer, probe oligomer); OL is independently at each occurrence absent or a sequence of nucleic acid residues (e.g., of from 1-10 bases in length); and CS2 is a complementary sequence of CS2

[0170] Non-limiting illustrative embodiment 31. The immunoassay composition according to any one of Non-limiting illustrative embodiments 28-30, wherein said composition further comprises a buffer.

[0171] Non-limiting illustrative embodiment 32. A solid particle coated with streptavidin conjugated to a biotinylated antibody for an analyte optionally through a linker (e.g., PEG, lodo-PEG), wherein the antibody is bound to the analyte, and the bound analyte is further bound to a second antibody (e.g., monoclonal mouse antibody) or fragment thereof of the analyte, wherein the second antibody is conjugated to a pre-amplifier oligomer, the pre-amplifier oligomer is hybridized to one or more amplifier oligomers; and the amplifier oligomer is hybridized to one or more probe labels comprising a chemiluminescent moiety (e.g., chemiluminescent acridinium) conjugated to a probe oligomer.

[0172] Non-limiting illustrative embodiment 33. The solid particle according Non-limiting illustrative embodiment 32, wherein the preamplifier oligomer may have the structure: 5 -TS-OL-(OL-C S1 -OL)m-0L-TS-3 ’ the amplifier oligomer may have the structure: S’-TS-OL-CS^-OL-tOL-CSVOL-TS-S’ the probe oligomer may have the structure: 5’-TS-CS2*-TS-3’ where m and n are independently from 1-20 (e.g., 2-10, 2-5) TS is independently at each occurence absent or a sequence of nucleic acid residues (e.g., of from 1-15 bases in length) and may cap the indicated oligomer (e.g., preamplifier oligomer, amplifier oligomer, probe oligomer); OL is independently at each occurrence absent or a sequence of nucleic acid residues (e.g., of from 1-10 bases in length); CS1 is a complementary sequence of CS1*; and CS2 is a complementary sequence of CS2*.

[0173] Non-limiting illustrative embodiment 34. A kit comprising two or more immunoassay reagents selected from a preamplifier reagent, a lite reagent, an amplifier reagent, and a solid phase reagent; wherein the preamplifier reagent comprises a preamplifer oligomer conjugated to a first binding partner for an analyte (e.g., antibody or antibody fragment) and a carrier or expcipient, the lite reagent comprises a probe oligomer bound to a chemiluminescent moiety and optionally and amplifier oligomer and a carrier or excipient, the amplifier reagent comprises an amplifier oligomer and a carrier or excipient, and the solid phase reagent comprises a particle having a second binding partner for the analyte (e.g., antibody or antibody fragment) attached to the surface of the particle and a carrier or excipient. Non-limiting illustrative embodiment 35. The kit according to Non-limiting illustrative embodiment 34, wherein the kit is used in the method according to any one of Non-limiting illustrative embodiments 1-27.

[0174] All references including patent applications and publications cited herein are incorporated herein by reference and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

1. A method for the detection or quantification of an analyte in a sample (e.g., a biological sample such as blood, saliva, serum, a sample derived from a biological sample such as a diluted biological sample) comprising:(a) mixing the sample with a composition comprising a branched DNA (b-DNA) pre-amplifier oligomer conjugated to a first antibody or antibody fragment that binds to the analyte and optionally a particle having a second antibody or antibody fragment that binds to the analyte attached to the surface of the particle;(b) adding to the mixture a particle having a second antibody or antibody fragment that binds to the analyte attached to the surface of the particle (e.g., if the particle is not already present in the mixture);(c) adding to the mixture a b-DNA amplifier oligomer that binds to the preamplifier oligomer, wherein, optionally, multiple amplifier oligomers bind to at least one of the pre-amplifier oligomers conjugated to the analyte;(d) adding to the mixture a label probe comprising a chemiluminescent moiety bound to a probe oligomer, wherein the probe oligomer binds one of the amplifier oligomers and, optionally, multiple probe oligomers bind to the same amplifier oligomer;(c) preparing the mixture to measure chemiluminescence (e.g., by separation of the particle having chemiluminescent acridiniums conjugated to the surface from the mixture);(d)    triggering chemiluminescence from the prepared mixture; and(e)    detecting the presence or calculating the concentration of said at least oneanalyte by comparing the amount of chemiluminescence with a standard dose response curve which relates the amount of light emitted to a know n concentration of the at least one of the multiple analytes.

2. The method according to claim 1, the analyte is an exosome (e.g., neuron-derived, astrocyte-derived, oligodendrocyte-derived, microglia-derived) protein biomarker.

3. The method according to claim 1 or 2, wherein the analyte is glypican-1 (GPC1), neurofilament light chain (NFL), phosphorylated tau (p-tau), or amyloid beta (AP).

4. The method according to any one of claims 1-3, wherein the probe oligomer is a an oligonucleotide comprising the sequence5-AGT FAJ CGC FGT AFT T-3£ (SEQ ID NO 9), or5 -GCA CTT GGT ACG GCG CTG ACT TTT-AE-3‘ (SEQ ID NO 12)or a sequence having more than 80% (e.g.. more than 90%, more than 95%) identity therewith; wherein F is isocytosine and J is isoguanine.

5. The method according to any one of claims 1-4, wherein the amplifier oligomer is an oligonucleotide comprising the sequence5'-TTT-(JTA CJG CGF TJA CT TTTTT)„-TCF ACG JCF CTA JG-3' (SEQ ID NO 10) or5‘ -TGA CAC GGG TCC TAT GCC TTT T(AG TCA GCG CCG TAC CAA GTG CTT TTT T)n -3' (SEQ ID NO 13)or a sequence having more than 80% (e.g.. more than 90%, more than 95%) identity therewith; wherein n is an integer (e.g., an integer from 1-50 or 1-20 or 2-50 or 2-20 or 2-15 or 5-15): F is isocytosine and J is isoguanine.

6. The method according to any one of claims 1-5. wherein the pre-amplifier oligomer is an oligonucleotide having the sequence5'-TTT-(CFT AGJ GFC GTJ GA TTTTT)n-3' (SEQ ID NO 11) or5 - AGG CAT AGG ACC CGT GTC TTT TTT (AGG CAT AGG ACC CGT GTC CGT GGA TGT TTG AGG CAT AGG ACC CGT GTC TTT TTT)n -3' (SEQ ID NO 14)or a sequence having more than 80% (e.g., more than 90%, more than 95%) identity therewith; wherein n is an integer (e.g.. an integer from 1-50 or 1-20 or 2-50 or 2-20 or 2-15 or 5-15): F is isocytosine and J is isoguanine.

7. The method according to any one of claims 1-6, wherein the label probe is added inmolar excess of pre-amplifier oligomer (e.g., less than 100x excess or less than 50* excess or less than 40* excess or less than 30x excess or from 5x excess to 25x excess).

8. The method according to any one of claims 1-7, wherein the chemiluminescent moiety is an acridium or an alkaline phosphatase.

9. The method according to any one of claims 1 -8, wherein the label probe was formed byi) reacting a chemiluminescent acridinium compound comprising a reactive functional group with a linking compound (e.g., a compound comprising a linker with reactive functional groups on each end, a compound already conjugated to the probe oligomer) andii) reacting the probe oligomer to the linking compound.

10. The method according to claim 9, wherein the chemiluminescent acridinium compoundcomprising a reactive functional group has the structure:RFGLT(I)wherein RFG is a reactive functional group for conjugation to the probe oligomer, L is absent (i.e., it is a bond) or a linker optionally comprising a group Lc or ZL, and T is a chemiluminescent acridinium comprising the structure:and are independently 0 (e.g, all R2 groups are hydrogen, all R3 groups are hydrogen), 1,2, 3, or 4;Ri is hydrogen, -R, -Xb, -RL-Xb. -Lc-R, -Lc-Xb (e.g, -Li-Xb), -Z, -RL-Z, -Lc-Z (e.g., -Li-Z), or -Rl-Lc-Rl-Z (e.g.. -Rl-Li-Rl-Z);R2 and R3 are independently selected at each occurrence from hydrogen. -R, an electron donating group, -Xc, -RL-XC, -Lc-Xc (e.g., -Li-Xc), and -Z; wherein two vicinal R2 or R? groups may together form a fused cyclic group (e.g., 5-7 membered fused aryl or heteroarylgroup, 5-7 membered fused heterocyclic group) and wherein R2 or R3 may comprise a linkage to an imaging agent such as a fluorophore (e.g., rhodamine);Lc is a divalent C1-35 alky l, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted (e.g., with 1 to 20 heteroatoms, with 1-20 substituents);ZL is a zwitterionic linker group having the structure:“»T is 0 (i.e. it is a bond) or 1;“«’■ and “p” are independently at each occurrence an integer from 0 (i.e. it is a bond) to 10;Z is a zwitterionic group independently at each occurrence has the structure:“9” and are independently 0 or 1;“r” is independently an integer from 0 to 10 (e.g., from 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);Xa and Xb are independently at each occurrence an anionic group;Xc is a protonated anionic group;Li is independently at each occurrence -O-, -S-, -NH-, -N(Rn)-, -(CH2)i-io-, -S(=O)i-2-, -C=C- -C=C-(CH2) 1-3-, -C(O)-. -O-C(O)-. -C(O)-(CH2) 1-4-, -(CH2)i-4-C(O)-, -C(O)-O-,    -C(O)-N(RN)-,    -C(O)-NH-,    -N(Rn)-C(O)-,    -NH-C(O)-,-C(O)-N(Rn)-(CH2)i-3-,       -(CH2)i-3-C(O)-N(Rn)-       -(CH2)i-3-N(Rn)-C(O)-,-NH-S(O)i-2-, -N(Rn)-S(O)i-2-, -S(O)i-2-N(Rn)- -S(O)i-2-NH-, -(CH2)i-3-NH-S(O)i-2--(CH2)i-3-N(Rn)-S(O)i-2-,      -(CH2)i-3-S(O)i-2-N(Rn)-      -(CH2)i-3-S(O)i-2-NH--O-(CH2)i-4-    -(CH2)i-4-O-,    -S-(CH2)i-4-    -(CH2)i-4-S-,    -NH-(CH2)i-4--N(Rn)-(CH2) 1-4-, -(CH2)i-4-N(Rn)- -(OCH2)i-10- -(CH20)i-io-, -(OCH2CH2)i-io-, or -(CH2CH20)i-io-;RLis independently at each occurrence a C1-20 bivalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, phenyl, mono alkyl substituted phenyl, di alkyl substituted phenyl, alkynyl. arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms, with 1-10 substituents);R is independently at each occurrence hydrogen or C1-35 hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms, with 1-20 substituents);R’ and R” are independently at each occurrence hydrogen or a C1-10 alkyl;RN is independently at each occurrence from hydrogen or C1-5 alkyl (e.g., methyl, ethyl, propyl); andR’ is hydrogen or a C1-10 alkyl; ora salt thereof (e.g., a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyd sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalky1 carboxylate salt).

11. The method according to any one of claims 1-10, wherein the second antibody or antibody fragment is a biotinylated antibody or antibody fragment.

12. The method according to any one of claims 1-11, wherein the particle is streptavidin coated.

13. The method according to any one of claims 1-12, wherein said detecting step can detect a difference in concentration of less than (or from 0.005 pg / mL to) 5 pg / mL (e.g., less than 1 pg / mL, less than 0.5 pg / mL, less than 0.2 pg / mL, from 0.01-0.2 pg / mL).

14. The method according to any one of claims 1-13, wherein said preparing step comprises separating the particle from the mixture and the chemiluminescence is triggered from the particle or the separated mixture.

15. The method according to any one of claims 1-14, wherein the method further comprising a washing step between the mixing step and any of the adding steps.

16. The method according to any one of claims 1-15, wherein the preamplifier oligomer has the structure:5 -TS-OL-(OL-C S ^Opm-OL-TS-S ’the amplifier oligomer has the structure:5 -TS-OL-CS ^-OL-COL-CSVOL-TS-S ’the probe oligomer has the structure:5’ TS CS2* TS 3’where m and n are independently from 1-20 (e.g.. 2-10. 2-5)TS is independently at each occurence absent or a sequence of nucleic acid residues (e.g., of from 1-15 bases in length) and may cap the indicated oligomer (e.g., preamplifier oligomer, amplifier oligomer, probe oligomer);OL is independently at each occurrence absent or a sequence of nucleic acid residues (e.g., of from 1-10 bases in length);CS1 is a complementary sequence of CS1*; andCS2 is a complementary sequence of CS2*.

17. An immunoassay composition comprising a chemiluminescent label conjugated to a probe oligomer; and a carrier or excipient.

18. The immunoassay composition according to claim 17, wherein said composition further comprises a buffer.

19. A solid particle coated with streptavidin conjugated to a biotinylated antibody for an analyte optionally through a linker (e.g., PEG, lodo-PEG), wherein the antibody is bound to the analyte, and the bound analyte is further bound to a second antibody (e.g., monoclonal mouse antibody) or fragment thereof of the analyte, wherein the second antibody is conjugated to a pre-amplifier oligomer, the pre-amplifier oligomer is hybridized to one or more amplifier oligomers; and the amplifier oligomer is hybridized to one or more probe labels comprising a chemiluminescent moiety (e.g., chemiluminescent acridinium) conjugated to a probe oligomer.

20. The solid particle according to claim 19. wherein the preamplifier oligomer has the structure:5 -TS-OL-(OL-C S ^OL^-OL-TS-S ’the amplifier oligomer has the structure:5 -TS-OL-CS ^-OL-COL-CSVOL-TS-S ’the probe oligomer has the structure:5’ TS CS2* TS 3’where m and n are independently from 1-20 (e.g.. 2-10. 2-5)TS is independently at each occurence absent or a sequence of nucleic acid residues (e.g., of from 1-15 bases in length) and may cap the indicated oligomer (e.g., preamplifier oligomer, amplifier oligomer, probe oligomer);OL is independently at each occurrence absent or a sequence of nucleic acid residues (e.g., of from 1-10 bases in length);CS1 is a complementary sequence of CS1*; andCS2 is a complementary sequence of CS2*.

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