Beta-galactosidase-based immunoassays

Abstract

An enzyme donor (ED) fragment of a beta-galactosidase (beta-gal) is provided. The present invention relates to an ED fragment comprising an amino acid sequence having at least 90% amino acid identity to the following amino acid sequence: CPGNIDPCASSNSLAVVLQRRDWENPGVTQL NRLAAHPPFASWRNSEEARTDD CPSQQLCQ (SEQ ID NO: 1) and comprising: i) cysteine (C) residues located at positions 1, 8, 53 and 59; ii) a C and acetylated N-terminus located at position 8; iii) C located in positions 1, 8 and 53; iv) C and acetylated N termini located at the 8th and 53rd positions; or v) a C and acetylated N-terminus located at position 53, wherein the residue position refers to the residue position in SEQ ID NO: 1.

Description

[0001] Cross-reference to related applications This application claims the benefit of U.S. Provisional Patent Application 63 / 390,544, filed July 19, 2022, which is incorporated herein by reference in its entirety.

[0002] introduction The analysis of biological samples often involves determining the presence of a target analyte in the sample. Target analytes may be present in trace amounts, requiring sensitive assays for detection. In many applications, in addition to sensitivity, rapid and easy access to analytical results is also desirable.

[0003] Complementation assays based on β-galactosidase (β-gal) have been used to detect target analytes. These assays typically measure the binding process of the enzyme donor (ED) fragment of β-gal with the enzyme acceptor (EA) fragment to form an enzymatically active β-gal. Summary of the Invention

[0004] An enzyme donor (ED) fragment for β-galactosidase (β-gal) is provided. The ED fragment contains an amino acid sequence with at least 90% amino acid identity to the following amino acid sequences: CPGNIDCASNSLAVVLQRRRDWENPGPTVQLNRLAAHPPFASWRNSEARTDCPSQQLCQ (SEQ ID NO: 1), and It includes: i) Cysteine ​​(C) residues located at positions 1, 8, 53, and 59; ii) The C at position 8 and the acetylated N-terminus; iii) C located at positions 1, 8, and 53; iv) The C at positions 8 and 53 and the acetylated N-terminus; or v) The C at position 53 and the acetylated N-terminus, The residue positions are referenced from those in SEQ ID NO: 1.

[0005] In some respects, the amino acid sequence of the ED fragment has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity with SEQ ID NO: 1. In some respects, the ED fragment has a length of up to 200 amino acids, up to 150 amino acids, up to 125 amino acids, up to 100 amino acids, up to 90 amino acids, up to 85 amino acids, up to 80 amino acids, up to 75 amino acids, up to 70 amino acids, up to 65 amino acids, or up to 60 amino acids.

[0006] In some respects, the amino acid sequence of the ED fragment contains i) cysteine ​​(C) residues at positions 1, 8, 53, and 59 and an acetylated N-terminus. In other respects, the ED fragment contains only four C residues.

[0007] In some respects, the amino acid sequence of the ED fragment includes ii) a C at position 8 and an acetylated N-terminus. In some respects, the ED fragment contains only one C residue.

[0008] In some respects, the amino acid sequence of the ED fragment includes iii) C residues at positions 1, 8, and 53 and an acetylated N-terminus. In other respects, the ED fragment contains only three C residues.

[0009] In some respects, the amino acid sequence of the ED fragment contains (iv) C residues at positions 8 and 53 and an acetylated N-terminus. In other respects, the ED fragment contains only two C residues.

[0010] In some respects, the amino acid sequence of the ED fragment includes a C at position 53 and an acetylated N-terminus. In other respects, the ED fragment contains only one C residue.

[0011] In some respects, the C-terminus of the amino acid sequence of the ED fragment contains an amide modification.

[0012] In some respects, the ED can be conjugated with a drug and can bind to the enzyme receptor (EA) fragment of β-galactosidase to form a catalytically active β-galase.

[0013] In some aspects, the drug is selected from: fentanyl, heroin, amphetamine, 3,4-methylenedioxyamphetamine (MDA), barbiturate, benzodiazepine, cocaine, benzoyl lecgonine, buprenorphine, cannabinoid, methadone, hydrocodone, ethanol, ethyl glucuronide, ketamine, meperidine, methamphetamine, opioids, oxycodone, morphine, or metabolites thereof. In some aspects, the drug is an MDA. In some aspects, the drug is cocaine or benzoyl lecgonine.

[0014] This disclosure also provides a kit comprising the ED and EA fragments disclosed herein, wherein the ED and EA fragments bind to form a catalytically active β-gal enzyme. The kit may further comprise an antibody that specifically binds to a drug. In some aspects, the ED fragment is conjugated to a drug. The drug may be selected from: fentanyl, heroin, amphetamine, 3,4-methylenedioxyamphetamine (MDA), barbiturates, benzodiazepines, cocaine, benzoyl methylphenidate, buprenorphine, cannabinoids, methadone, hydrocodone, ethanol, ethyl glucuronide, ketamine, meperidine, methamphetamine, opioids, oxycodone, morphine, or metabolites thereof. The kit may further comprise a substrate for the β-gal enzyme.

[0015] This document also discloses a method for detecting a drug in a sample. The method may include combining: the ED fragment disclosed herein (wherein the ED fragment is conjugated to a drug), an enzyme receptor (EA) fragment of a β-galase, an antibody that specifically binds to the drug, a sample, and a β-galase substrate; and measuring a signal indicating the reconstitution of an active β-galase, wherein the presence of the signal indicating the reconstitution of an active β-galase indicates the presence of a drug in the sample, and wherein the absence of the signal indicating the reconstitution of an active β-galase indicates the absence of a drug in the sample.

[0016] In some respects, the drug is selected from: fentanyl, heroin, amphetamine, 3,4-methylenedioxyamphetamine (MDA), barbiturates, benzodiazepines, cocaine, benzoyl styrene, buprenorphine, cannabinoids, methadone, hydrocodone, ethanol, ethyl glucuronide, ketamine, pethidine, methamphetamine, opioids, oxycodone, morphine, or their metabolites. Attached Figure Description

[0017] Figure 1 An exemplary assay for detecting a drug in a sample is described. In this assay, a sample suspected of containing a drug is combined with an antibody that binds to the drug, a β-gal enzyme receptor (EA) fragment, and a β-gal enzyme donor (ED) fragment conjugated to the drug (ED-drug). If the drug is not present in the sample, the antibody binds to the ED-drug, thereby isolating the ED-drug and preventing it from binding to the EA. No signal is detected upon addition of a β-gal substrate, indicating the absence of a detectable level of drug in the sample. If the drug is present in the sample, the antibody binds to the drug, allowing the ED-drug to bind to the EA fragment to form a catalytically active β-gal enzyme, which is detectable upon addition of a β-gal substrate. The presence of a signal indicates the presence of the drug in the sample.

[0018] Figure 2A schematic diagram illustrating the modifications made to the ED fragment is shown. “SH” indicates the presence of cysteine ​​residues. “NH2” indicates amination.

[0019] Figure 3 The graph shows the signal (Y-axis) indicating the presence of 3,4-methylenedioxyamphetamine (MDA) at the concentrations shown on the X-axis. MDA was detected using the standard ED60-MDA conjugate, the ED60-MDA conjugate complexed with an additional MDA molecule, and the ED fragment mutant D-MDA.

[0020] Figure 4 A graph indicating the signal (Y-axis) of hydrocodone present at the concentrations shown on the X-axis is displayed. Hydrocodone was detected using the standard ED60-hydrocodone conjugate and the ED fragment mutant D-hydrocodone.

[0021] Figure 5 The graph shows the signal (Y-axis) indicating the presence of benzoyl sphagnum at the concentrations shown on the X-axis. Benzoyl sphagnum was detected using the standard ED60-benzoyl sphagnum conjugate and the ED fragment mutant D-benzoyl sphagnum. Detailed Implementation

[0022] A novel polypeptide is provided that serves as an enzyme donor (ED) fragment in a complex containing an enzyme acceptor (EA) fragment of β-galactosidase. The ED fragment comprises an amino acid sequence having at least 90% amino acid identity with the following amino acid sequence: CPGNIDCASNSLAVVLQRRRDWENPGPTVQLNRLAAHPPFASWRNSEARTDCPSQQLCQ (SEQ ID NO: 1), and It includes: i) Cysteine ​​(C) residues located at positions 1, 8, 53, and 59; ii) The C at position 8 and the acetylated N-terminus; iii) C located at positions 1, 8, and 53; iv) The C at positions 8 and 53 and the acetylated N-terminus; or v) The C at position 53 and the acetylated N-terminus, The residue positions are referenced from those in SEQ ID NO: 1.

[0023] Before describing this disclosure in more detail, it should be understood that this disclosure is not limited to the specific embodiments described, and therefore changes are naturally possible. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be restrictive.

[0024] When a numerical range is provided, it should be understood that, unless the context explicitly specifies otherwise, the numerical range includes every intermediate value between the upper and lower limits of the range (accurate to one-tenth of the lower limit unit), as well as any other specified value or intermediate value within the specified range.

[0025] Certain ranges presented in this document are preceded by the term "approximately". In this document, the term "approximately" is used to provide textual support for the exact figures preceding it and for figures that are close to or approximate to the figures preceding the term. In determining whether a figure is close to or approximates an explicitly listed figure, an unlisted close or approximate figure may be a figure that, in its context, provides a substantial equivalent to the explicitly listed figure.

[0026] Unless otherwise defined, 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.

[0027] This disclosure can be more readily understood by referring to the following detailed description of the desired implementation and the embodiments it includes. In the following specification and subsequent claims, certain terms will be referenced and defined as having the following meanings.

[0028] Although specific terms are used in the following description for clarity, these terms are used only to refer to the specific structures of the selected embodiments illustrated in the accompanying drawings and are not intended to limit or restrict the scope of this disclosure. In the accompanying drawings and the subsequent description, it should be understood that similar numerical designations denote functionally similar components.

[0029] Unless the context clearly indicates otherwise, the singular forms “a”, “an”, and “the” include plural referents.

[0030] As used herein, the term "comprising" means that the specified component is required, but other components are permitted. The term "comprising" should be understood to include both the terms "substantially composed of" and "composed of". "Substantially composed of" permits the presence of the specified component, as well as other components that do not alter the function / structure of the specified component. "composed of" permits the presence of the specified component, as well as any adhesives or other bonding methods used to attach the listed components.

[0031] The numerical value should be understood to include the same numerical value when reduced to the same number of significant figures, as well as the numerical value whose difference from the value is less than the experimental error of the type of conventional measurement technique used to determine the value as described in this application.

[0032] All ranges disclosed herein include the stated endpoints and can be combined independently (e.g., the range “2 g to 10 g” includes the endpoints 2 g and 10 g, as well as all intermediate values). The range endpoints and any values ​​disclosed herein are not limited to precise ranges or values; they are sufficiently imprecise to include values ​​that approximate these ranges and / or values.

[0033] The modifier “about” used in relation to quantity includes the stated value and has a meaning determined by the context. When used in a context involving ranges, the modifier “about” should also be considered to disclose a range defined by the absolute values ​​of the two endpoints. For example, the range “about 2 to about 10” also discloses a range of “2 to 10”. The term “about” can refer to ±10% of the indicated number. For example, “about 10%” can represent a range of 9% to 11%, and “about 1” can represent a range of 0.9 to 1.1.

[0034] All publications and patents referenced in this specification are incorporated herein by reference as if each publication or patent were specifically and individually stated to be incorporated herein by reference, and the references are incorporated herein to disclose and describe methods and / or materials relating to the referenced publications. Any reference to a publication refers to its publication prior to the filing date and should not be construed as an admission that the invention is not entitled to a prior art invention prior to that publication. Furthermore, the publication dates provided may differ from the actual publication dates, which require separate verification.

[0035] It is worth noting that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” also include plural references, unless the context clearly specifies otherwise. It should also be noted that claims can be drafted to exclude any optional elements. Therefore, this statement is intended as a basis for the use of exclusive terms such as “only,” “only,” or the use of negative limiting terms in relation to the description of claim elements.

[0036] Upon reading this disclosure, it will be apparent to those skilled in the art that the various embodiments described and illustrated herein have discrete components and features, which can be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of the invention. Any of the listed methods may be performed in the order of the events listed or in any other logically feasible order.

[0037] β-GAL enzyme donor fragment As summarized above, aspects of this disclosure include ED fragments comprising an amino acid sequence having at least 90% amino acid identity with the following amino acid sequence: CPGNIDCASNSLAVVLQRRRDWENPGPTVQLNRLAAHPPFASWRNSEARTDCPSQQLCQ (SEQ ID NO: 1), and It includes: i) Cysteine ​​(C) residues located at positions 1, 8, 53, and 59; ii) The C at position 8 and the acetylated N-terminus; iii) C located at positions 1, 8, and 53; iv) The C at positions 8 and 53 and the acetylated N-terminus; or v) The C at position 53 and the acetylated N-terminus, The residue positions are referenced from those in SEQ ID NO: 1.

[0038] In some respects, the amino acid sequence of the ED fragment has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity with SEQ ID NO: 1. In some respects, the amino acid sequence of the ED fragment includes conserved substitutions relative to the amino acid sequence of SEQ ID NO: 1. In most cases, conserved substitutions are involved, wherein nonpolar aliphatic amino acids (e.g., G, A, V, L, and I) may substitute for each other; uncharged polar amino acids (e.g., C, M, S, T, N, and Q) may substitute for each other; charged amino acids may substitute for each other with the same charge, i.e., K and R; and D and E; and aromatic amino acids F, W, and Y may substitute for each other.

[0039] In some respects, the ED fragment has a length of up to 200 amino acids, up to 150 amino acids, up to 125 amino acids, up to 100 amino acids, up to 90 amino acids, up to 85 amino acids, up to 80 amino acids, up to 75 amino acids, up to 70 amino acids, up to 65 amino acids, or up to 60 amino acids. In some respects, the amino acid sequence of the ED fragment may be reduced by up to 1, 2, 3, 4, 5, or 6 amino acids relative to SEQ ID NO: 1. In some respects, the amino acid sequence of the ED fragment may be increased by up to 5, 10, 20, 30, 40, 50, or 60 amino acids relative to SEQ ID NO: 1, wherein the added amino acids are identical to those present at the corresponding positions in naturally occurring β-galases.

[0040] In some respects, the amino acid sequence of the ED fragment contains i) cysteine ​​(C) residues at positions 1, 8, 53, and 59 and an acetylated N-terminus. In some respects, the ED fragment contains only four C residues. In some respects, the amino acid sequence of the ED fragment is as shown in SEQ ID NO: 1. In some respects, the ED fragment is acetylated at the N-terminus and contains an amide at the C-terminus.

[0041] In some respects, the amino acid sequence of the ED fragment includes ii) a C at position 8 and an acetylated N-terminus. In some respects, the ED fragment contains only one C residue. In some respects, the amino acid sequence of the ED fragment includes the amino acid sequence shown in SEQ ID NO: 5. In some respects, the ED fragment is acetylated at the N-terminus and contains an amide at the C-terminus.

[0042] In some respects, the amino acid sequence of the ED fragment contains iii) C residues at positions 1, 8, and 53 and an acetylated N-terminus. In some respects, the ED fragment contains only three C residues. In some respects, the amino acid sequence of the ED fragment contains the amino acid sequence shown in SEQ ID NO: 6. In some respects, the ED fragment is acetylated at the N-terminus and contains an amide at the C-terminus.

[0043] In some respects, the amino acid sequence of the ED fragment contains iv) C residues at positions 8 and 53 and an acetylated N-terminus. In some respects, the ED fragment contains only two C residues. In some respects, the amino acid sequence of the ED fragment contains the amino acid sequence shown in SEQ ID NO:7. In some respects, the ED fragment is acetylated at the N-terminus and contains an amide at the C-terminus.

[0044] In some respects, the amino acid sequence of the ED fragment contains v) a C at position 53 and an acetylated N-terminus. In some respects, the ED fragment contains only one C residue. In some respects, the amino acid sequence of the ED fragment contains the amino acid sequence shown in SEQ ID NO: 8. In some respects, the ED fragment is acetylated at the N-terminus and contains an amide at the C-terminus.

[0045] As used in this article, "amino" refers to -NH2, "carboxyl" refers to -CO2H, and "acetyl" refers to -C(O)CH3. Protein Nt-acetylation refers to the covalent attachment of an acetyl group (CH3CO) to the free α-amino group (NH3) at the N-terminus of a polypeptide. + C-terminal amidation removes the charge from the C-terminus of the peptide. C-terminal amidation of the ED fragments disclosed herein may include attaching an amide (NH2) or an N-alkylamide to the C-terminus. "alkyl" refers to a saturated, straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, having 1 to 12 carbon atoms (C1-C2). 12 Alkyl group, having 1 to 8 carbon atoms (C1-C8 alkyl) or 1 to 6 carbon atoms (C1-C6 alkyl), and attached to the rest of the molecule by a single bond.

[0046] The ED can be linked to any target compound. The ED of the present invention can be used for both competitive and non-competitive assays to detect the presence of drugs, peptides (e.g., antibodies), lectins, nucleic acids, sugars, or other target analytes. Examples of using EDs to determine analytes can be found, for example, in U.S. Patent Nos. 4,378,428, 4,708,929, 5,037,735, 5,106,950, 5,362,625, 5,464,747, 5,604,091, and 5,643,734, all of which are specifically incorporated by reference to illustrate how these assays are performed, wherein the ED of the present invention may directly replace the ED used in the illustrated references.

[0047] EDs can be prepared by any convenient means. When EDs are linked to substances other than peptides, they can be synthesized using conventional methods with commercially available synthesizers, or they can be prepared using cloning, in which case the ED can be generated intracellularly and isolated by lysis, or secreted using an appropriate signal sequence. However, when fusion proteins are used and the fusion protein substantially exceeds about 60 amino acids, cloning is usually employed, in which case the protein can be isolated by lysis or by secretion from the culture medium as described above.

[0048] ED fragments can be synthesized on an automated peptide synthesizer using standard synthetic techniques. In some cases, protected amino acids representing the carboxyl-terminal amino acid of the desired peptide are attached to cross-linked polystyrene beads. The resin beads act as a solid phase, allowing additional amino acids to be coupled to the solid phase in a stepwise manner. The peptide is generated by sequentially elongating the chain from the carboxyl-terminus to the N-terminus. The solid phase facilitates rapid driving of the reaction to 100% completion using excess reagent. The excess reagent can then be easily washed away. After the synthetic steps are complete, the peptide is removed from the resin and purified.

[0049] ED-drug conjugates In some respects, ED can conjugate with drugs and can bind to the enzyme receptor (EA) fragment of β-galactosidase to form a catalytically active β-galase.

[0050] In some respects, the drugs are selected from: fentanyl, heroin, amphetamine, 3,4-methylenedioxyamphetamine (MDA), barbiturates, secobarbital, diazepam, oxazepam, benzodiazepines, cocaine, benzoyl methionine, buprenorphine, cannabinoids, methadone, hydrocodone, ethanol, ethyl glucuronide, ketamine, meperidine, methamphetamine, opioids, oxycodone, morphine, quinidine, propoxyphen, N-acetylprocainamide. Procainamide, tobramycin or its derivatives (e.g., metabolites), estriol, digoxin, thyroxine, propranolol, methotrexate, phencyclidine, methadone, gentamicin, theophylline, benzoyl benzoate, phenytoin, procainamide, lidocaine, carbamazepine, primidone, valproic acid, phenobarbital, ethosuximide, biotin or its metabolites or derivatives. In some aspects, the drug is an MDA. In some aspects, the drug is cocaine or benzoyl benzoate.

[0051] The ED fragments disclosed herein include introduced amino acids, such as cysteine ​​or lysine, for coupling the analyte to the ED fragment. When a free thiol group is present, it can react with a reactive group present on the analyte. Such reactive groups include, but are not limited to, reactive haloalkyl groups and acid / halogen groups, p-mecuribenzoate groups, and groups capable of Michael-type addition reactions (including, for example, maleimide and the group types described in Mitral and Lawton, 1979, J. Amer. Chem. Soc. 101:3097-3110). Haloalkyl groups as defined herein include any alkyl group with one to three carbon atoms substituted by bromine, iodine, or chlorine. If the analyte does not have such a reactive group for coupling to the enzyme donor with a free thiol group, an analyte derivative containing such a reactive group can be prepared.

[0052] ED-peptide fusion In some respects, ED fragments can conjugate with heterologous peptides. As used herein, the term "heterologous" refers to a component of a peptide that is not native to the peptide in question, i.e., one that is typically found in nature and does not bind to the remainder of the peptide being compared.

[0053] According to another embodiment of the invention, an enzyme donor polypeptide is prepared by linking or fusing a gene encoding an ED with another gene encoding a heterologous protein analyte (or a portion thereof). The linked gene is expressed in a suitable host cell, producing a fusion protein product that is both complementary to the enzyme receptor and specifically binds to an analyte-binding protein (e.g., a receptor or antibody). Therefore, the fusion protein prepared according to this embodiment of the invention comprises two domains: (1) an ED domain and (2) a protein domain, both encoded by the fusion gene. The spacer sequence between the ED and protein domains can be used to enhance complementarity.

[0054] The analytes may be human glycoproteins such as leutropin, leutinizing hormone, follicle-stimulating hormone, thyroid-stimulating hormone, thyroid-stimulating hormone, or human chorionic gonadotropin (hCG), carcinoembryonic antigen, ferritin, human T-cell leukemia virus, insulin, alpha-fetoprotein, herpesvirus, cytomegalovirus, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, hepatitis virus, human chorionic gonadotropin, estrogen receptor, thyroid-stimulating hormone receptor, poliovirus receptor, insulin transporter, protein A, concanavalin A, wheat germ agglutinin, secretory protein, cholera toxin, or avidin.

[0055] ED-analyte fusions can be used in combination with EA and peptides (such as antibodies or receptors) that bind to the analyte to detect the presence of the analyte in biological samples.

[0056] EA fragments β-galactosidase is a tetrameric protein with a molecular weight (MW) of 540,000 Daltons. It consists of four identical monomers, each containing 1,021 amino acids and 116,000 Daltons of MW. The monomeric protein is divided into three regions: (1) the proximal N-terminal region (α region), (2) the intermediate region, and (3) the distal C-terminal region (ω region).

[0057] In some respects, the EA fragment used in the methods and kits disclosed herein contains the sequence of the enzyme receptor (also known as the ω domain) of β-galactosidase, which is a larger fragment of β-galactosidase. This fragment can bind to the ED fragment disclosed herein and reconstitute active β-galactosidase. The EA fragment is available from commercial suppliers.

[0058] Substrate In some respects, ED is used in conjunction with EA to form an active β-galactosidase, which can be detected by adding a detectable substrate (typically a colored, fluorescent, or chemiluminescent substrate). β-galactosidases efficiently utilize fluorophores with phenolic groups that are etherified with the β-galactosyl group. Common substrates are β-D-galactopyranosylphenol, such as monosubstituted and disubstituted fluorescein, o-nitrophenyl-β-D-galactoside, β-methylumbelliferyl-β-D-galactoside, X-gal, halogen-β-D-galactoside, commercially available oxetanes (e.g., the Galacto-Light Plus® kit (chemiluminescent)), and chlorophenol red. Of particular interest are β-galactosidase substrates (especially due to their suitability for spectrophotometric or fluorescence analysis) including, but not limited to: p-aminophenyl-β-D-galactopyranoside; 2'-N-(hexadecyl)-N-(amino-4'-nitrophenyl)-β-D-galactopyranoside; 4-methylumbelliferyl-β-D-galactopyranoside; naphthyl-AS-Bl-β-D-galactopyranoside; 1-naphthyl-β-D-galactopyranoside; 2-naphthyl-β-D-pyranoside. Galactoside monohydrate; O-nitrophenyl-β-D-galactopyranoside; m-nitrophenyl-β-D-galactopyranoside; p-nitrophenyl-β-D-galactopyranoside; and phenyl-β-D-galactopyranoside, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, halogen-β-D-galactopyranoside, 7-hydroxy-4-trifluoromethylcoumarin, ω-nitrostyryl-β-D-galactopyranoside and fluorescein-β-D-galactopyranoside.

[0059] Enzyme complementation assay The amount of an unknown analyte in a sample mixture can be measured as a direct function of β-galactosidase activity. Enzyme activity is monitored by the appearance of enzyme-catalyzed reaction products or by the disappearance of enzyme substrates. This is the substrate conversion rate.

[0060] Advantageously, the assay disclosed herein is a homogeneous assay, eliminating the need to separate unbound components, unconverted substrates, or purify reaction products. Such assays are easily automated.

[0061] Enzyme activity can be measured by monitoring the substrate conversion rate of an enzyme-catalyzed reaction using any of a variety of techniques, including but not limited to spectrophotometry and fluorescence assays.

[0062] In some respects, enzyme complementation assays are immunoassays. Immunoassays may involve ED-analyte conjugates, EA, analyte-binding molecules (e.g., antibodies, such as monoclonal or polyclonal antibodies), and β-gal substrates. If the analyte is present in the sample, the analyte competes with the ED-analyte conjugate for binding to the analyte-binding molecule, resulting in an increased amount of the ED-analyte conjugate that can bind to EA to form an enzymatically active β-gal.

[0063] In some respects, methods for detecting the presence and optional concentration of an analyte in a sample may involve combining the following: the ED fragment disclosed herein (wherein the ED fragment is conjugated to an analyte), the enzyme receptor (EA) fragment of a β-galase, a polypeptide (e.g., an antibody) that specifically binds to the analyte, the sample, and a β-galase substrate. This combination may be performed in a single step, or any number of individual components may be premixed prior to combination with the sample and / or substrate. The combined mixture may optionally be incubated for any suitable period of time at room temperature, 4°C, 30°C, 37°C, or other suitable temperatures.

[0064] The method may further include measuring a signal indicating the reconstitution of active β-gal enzyme, wherein the presence of the signal indicating the reconstitution of active β-gal enzyme indicates the presence of a drug in the sample, and the absence of the signal indicating the reconstitution of active β-gal enzyme indicates the absence of a drug in the sample.

[0065] Analytes The analytical device (ED) of this invention can be used to determine the presence and / or amount of a variety of analytes, including drugs and drug metabolites, bioactive molecules, steroids, vitamins, industrial contaminants, pesticides and their metabolites, food additives, herbicides and their metabolites, flavorings and food toxins, pathogens and their toxins, and other target substances. Relatively high molecular weight analytes (e.g., proteins with a molecular weight greater than about 2,000 Daltons) as well as smaller analytes can be detected and / or measured using the disclosed ED. Exemplary examples of such analytes include, but are not limited to, the following drugs: fentanyl, heroin, amphetamines, 3,4-methylenedioxyamphetamine (MDA), barbiturates, secobarbital, diazepam, oxazepam, benzodiazepines, cocaine, benzoyl methionine, buprenorphine, cannabinoids, methadone, hydrocodone, ethanol, ethyl glucuronide, ketamine, meperidine, methamphetamine, opioids, hydroxychloroquine. Ketones, morphine, quinidine, propoxyphene, N-acetylprocainamide, tobramycin or its derivatives (e.g., metabolites), estriol, digoxin, thyroxine, propranolol, methotrexate, phencyclidine, methadone, gentamicin, theophylline, benzoyl succinate, phenytoin, procainamide, lidocaine, carbamazepine, primidone, valproic acid, phenobarbital, ethosuximide, biotin or its metabolites or derivatives.

[0066] In some respects, the analytes are selected from carcinoembryonic antigen, ferritin, human T-cell leukemia virus, insulin, alpha-fetoprotein, herpesvirus, cytomegalovirus, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, hepatitis virus, human chorionic gonadotropin, estrogen receptor, thyroid-stimulating hormone receptor, poliovirus receptor, insulin transporter, protein A, concanavalin A, wheat germ agglutinin, secretory protein, cholera toxin, or avidin.

[0067] Reagent test kit The components of the enzyme complementation assay of this invention can be packaged in a kit, in an aqueous medium, or in lyophilized form. Each component or reagent can be packaged individually or together with other components, provided that the sensitivity of the assay is not altered and the component is not adversely affected. The aqueous medium may contain buffers, protease inhibitors, and / or preservatives. In some respects, the substrate may be placed in an opaque container to avoid light exposure.

[0068] The kit may optionally include a positive control and / or a negative control. In some respects, the kit may contain multiple analytes at known concentrations for calibrating instruments used to detect signals generated during analyte assays and / or for generating concentration profiles of the analytes. Example

[0069] Immunosigma based on β-galactosidase The following enzyme donor fragments were generated and compared with AIA ED60. AIA ED60 contains two C residues. Table 1 shows the results:

[0070] Figure 1 A schematic diagram of a β-galactosidase-based immunoassay for detecting drugs in a sample is shown.

[0071] Figure 2 A schematic diagram of the tested ED60 segments is shown. ED60-D performed best. Further modifications to ED60-D are shown in Table 2:

[0072] As described below, ED and drug conjugation are combined according to Figure 1 The diagram shown is used for measurement.

[0073] Synthesis of MDA derivatives: Procedure: 100 mg of MDA HCl salt was dissolved in 20 ml of 5% sodium bicarbonate solution, extracted with ethyl acetate (3 × 20 ml), washed with water (3 × 30 ml), dried over sodium sulfate, filtered, and evaporated under vacuum to obtain 50 mg of MDA. MDA was dissolved in 2 ml of DMF at room temperature, and 98 mg (1.2 eq) of bromoacetylglycine NHS ester and 15 µl of DIPEA were added to the solution, respectively. The mixture was stirred at room temperature for 2 hours, diluted with 100 ml of ethyl acetate, washed with 20 ml of 5% sodium bicarbonate solution, washed with water (3 × 30 ml), dried over sodium sulfate, filtered, and evaporated under vacuum to obtain 75 mg of the product. MS: 357 (M+H+, 100%), 359 (M+2+H+, 98%).

[0074] Synthesis of BE or cocaine derivatives: Procedure: Benzoyl succinate (50 mg) was dissolved in DMF (1 ml) at room temperature; tributylamine (48 µl) and IBCF (22 µl, 1.2 eq) were added to the solution; the mixture was stirred at room temperature for 1 hour; N-(2-aminoethyl)maleimide TFA salt (48 mg) was added to the solution; the mixture was stirred at room temperature overnight; 1 ml of water was added, and the solvent was removed under reduced pressure; the mixture was packed into a pre-packed column; and purified using an ISCO column (MeOH / CH2Cl2 (0.1% Et3N)) to obtain 50 mg of the product. MS: 412 (M+H+).

[0075] Synthesis of hydrocodone derivatives: Hydromorphone HCl salt (200 mg) was suspended in benzene (3 ml). Ethylene glycol (2 ml) and TsOH (300 mg) were added to the suspension. The mixture was heated and refluxed for 30 minutes, then cooled to room temperature. 30 ml of 5% sodium bicarbonate was added to the solution. The mixture was extracted with ethyl acetate (3 × 20 ml), washed with water (3 × 30 ml), dried over sodium sulfate, filtered, and evaporated under vacuum to give 6-vinyl acetal-hydromorphone (white solid, 160 mg). MS: 330 (M+H+).

[0076] 6-Ethyleneacetal-hydromorphone (160 mg), PPh3 (0.5 g), and N-Boc-ethanolamine (0.3 g) were dissolved in anhydrous THF (20 ml) at room temperature under nitrogen protection; the solution was cooled to 0 °C in an ice-water bath. DIAD (0.4 g) was slowly added; the solution was stirred overnight at room temperature; the solvent was removed under reduced pressure; and the solution was purified using an ISCO column (EtOAc / MeOH 0.1% Et3N) to give 160 mg of 3-O-Boc-aminoethyl-6-ethyleneacetal-hydromorphone. MS: 473 (M+H+).

[0077] 3-O-Boc-aminoethyl-6-acetal-hydromorphone (160 mg) was dissolved in methanol (6 ml); 3N HCl (6 ml) was added to the solution; the mixture was heated and kept under reflux for 4 hours. All solvent was removed under vacuum; the solution was neutralized with 5% sodium bicarbonate solution, and the pH was adjusted to 8. The solvent was removed under vacuum; the solution was dissolved in acetonitrile (3 × 30 ml); the solution was filtered and evaporated under vacuum to give 3-O-aminoethyl-hydromorphone (100 mg). MS: 329 (M+H+).

[0078] 3-O-aminoethyl-hydromorphone (100 mg) was dissolved in 1 M sodium bicarbonate solution (5 ml); cooled to 0 °C in an ice-water bath, N-(methoxycarbonyl)maleimide (37 mg) was added to the solution; the mixture was stirred at this temperature for 30 minutes; 5 ml of acetonitrile was added, and the mixture was stirred at room temperature for 30 minutes. An additional 20 ml of acetonitrile was added to the turbid solution; the mixture was stirred and filtered; the solution was washed with ACN (5 ml); the pH was adjusted to 5 with 6 N HCl solution; the solvent was removed under vacuum; the solution was dissolved in ethanol (10 ml); the mixture was filtered and evaporated under vacuum to give hydromorphone-3-maleimide (100 mg). MS: 409 (M+H+).

[0079] Bioconjugation of β-galactosidase donor peptide mutant D with MDA-Br HPLC purification of MDA-Br derivatives: 1. Dissolve 1 mg of MDA-Br derivative in a mixture of 0.5 ml HPLC buffer B (0.1% TFA acetonitrile) and 0.7 ml HPLC buffer A (0.1% TFA water). 2. Inject 1.2 ml or 1 mg of the derivative solution into the HPLC. Waters HPLC (PDA996, ​​600 controller, binary pump). Phenomenex Kinetex® 5 µm XB C-18 100µm column (4.6*250 mm). HPLC method: Linear gradient: starting with 10% acetonitrile, then 40% acetonitrile at 30 min, followed by 100% acetonitrile. Flow rate: 1 ml / min. The peak at elution time 25.8 min is purified MDA-Br with correct mass spectrometry analysis. 3. Structure of MDA-Br derivatives

[0080] HPLC purification of peptide mutant D: 1. Dissolve 1 mg of peptide mutant D in 1 ml of HPLC buffer A (0.1% TFA water). 2. Inject 0.5 ml or 0.5 mg of the peptide mutant solution into the HPLC. Waters HPLC (PDA996, ​​600 controller, binary pump). Phenomenex Kinetex® 5 µm XB C-18 100µm column (4.6*250 mm). HPLC method: linear gradient elution: starting with 5% acetonitrile, then 45% acetonitrile at 35 min, followed by 100% acetonitrile. Flow rate: 1 ml / min. The peak at elution time 34.7 min represents the purified mutant D and will be used for the next conjugation step.

[0081] Preparation of MDA-Br mutant peptide D conjugate: 1. Mix 212 μL or 0.5 mg of peptide mutant HPLC fraction (elution time 34.7 min) with 510 μL or 1 mg of MDA-Br HPLC fraction (elution time 25.8 min). 2. Adjust the pH of the mixture to between 7.0 and 7.5 using 100 μL of 0.1 M phosphate buffer (pH 8.0) and an appropriate amount of 6 N NaOH. 3. Rotate at room temperature for 4 hours. 4. Conjugate HPLC Purification: Dissolve 400 µl of the conjugate mixture in 1 ml HPLC buffer A and inject it into the HPLC system. Waters HPLC (PDA996, ​​600 controller, binary pump). Phenomenex Kinetex® 5 µm XB C-18 100µl column (4.6*250 mm). HPLC Method: Linear gradient elution: starting with 10% acetonitrile, then 45% acetonitrile at 30 min, followed by 100% acetonitrile. Flow rate: 1 ml / min. The MDA-Br peptide mutant D conjugate showed a peak at 32.1 min, indicating correct immunoassay performance.

[0082] Figure 3-5 The results show the detection of various drugs using ED60.

[0083] Therefore, the foregoing only illustrates the principles of this disclosure. It should be understood that those skilled in the art will be able to design various arrangements that, although not explicitly described or shown in this invention, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language listed herein are primarily intended to help the reader understand the principles of the invention and the ideas contributed by the inventors to broaden the field, and should be interpreted as not being limited to these specifically listed examples and conditions. Moreover, all statements herein recounting the principles, aspects, and embodiments of the invention and their specific examples are intended to cover their structural and functional equivalents. Additionally, it is intended that such equivalents include currently known equivalents as well as those developed in the future, i.e., any element developed that performs the same function (regardless of its structure). Therefore, it is not intended that the scope of the invention be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied in the appended claims.

Claims

1. An enzyme donor (ED) fragment of a β-galactosidase (β-gal) comprising an amino acid sequence having at least 90% amino acid identity with the following amino acid sequence: CPGNIDCASNSLAVVLQRRRDWENPGPTVQLNRLAAHPPFASWRNSEARTDCPSQQLCQ (SEQ ID NO:1), and It includes: i) Cysteine ​​(C) residues located at positions 1, 8, 53, and 59; ii) The C at position 8 and the acetylated N-terminus; iii) C located at positions 1, 8, and 53; iv) The C at positions 8 and 53 and the acetylated N-terminus; or v) The C at position 53 and the acetylated N-terminus.

2. The ED fragment of claim 1, wherein the amino acid sequence has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity with SEQ ID NO:

1.

3. The ED fragment of claim 1, wherein the ED fragment has a length of up to 200 amino acids, up to 150 amino acids, up to 125 amino acids, up to 100 amino acids, up to 90 amino acids, up to 85 amino acids, up to 80 amino acids, up to 75 amino acids, up to 70 amino acids, up to 65 amino acids, or up to 60 amino acids.

4. The ED fragment of claim 1, wherein the amino acid sequence comprises i) cysteine ​​(C) residues at positions 1, 8, 53 and 59 and an acetylated N-terminus.

5. The ED fragment of claim 4, wherein the ED fragment contains only four C residues.

6. The ED fragment of claim 1, wherein the amino acid sequence comprises ii) a C at position 8 and an acetylated N-terminus.

7. The ED fragment of claim 6, wherein the ED fragment contains only one C residue.

8. The ED fragment of claim 1, wherein the amino acid sequence comprises iii) C and acetylated N-terminus located at positions 1, 8 and 53.

9. The ED fragment of claim 8, wherein the ED fragment contains only three C residues.

10. The ED fragment of claim 1, wherein the amino acid sequence comprises (iv) C and acetylated N-terminus located at positions 8 and 53.

11. The ED fragment of claim 10, wherein the ED fragment contains only two C residues.

12. The ED fragment of claim 1, wherein the amino acid sequence comprises v) a C at position 53 and an acetylated N-terminus.

13. The ED fragment of claim 12, wherein the ED fragment contains only one C residue.

14. The ED fragment of any one of claims 1 to 13, wherein the C-terminus comprises an amide modification.

15. The ED fragment of any one of claims 1-14, wherein the ED is conjugated with a drug and can bind to the enzyme receptor (EA) fragment of the β-galactosidase to form a catalytically active β-galase.

16. The ED fragment of claim 15, wherein the drug is selected from: fentanyl, heroin, amphetamine, 3,4-methylenedioxyamphetamine (MDA), barbiturates, benzodiazepines, cocaine, benzoyl styrene, buprenorphine, cannabinoids, methadone, hydrocodone, ethanol, ethyl glucuronide, ketamine, meperidine, methamphetamine, opioids, oxycodone, morphine, or metabolites thereof.

17. The ED fragment of claim 16, wherein the drug is MDA.

18. The ED fragment of claim 16, wherein the drug is cocaine or benzoyl succinate.

19. A reagent kit comprising: The ED fragment according to any one of claims 1-14; and The enzyme receptor (EA) fragment of β-galase.

20. The kit of claim 19, further comprising an antibody that specifically binds to the drug.

21. The kit of claim 20, wherein the ED fragment is conjugated with the drug.

22. The kit of claim 20, wherein the drug is selected from: fentanyl, heroin, amphetamine, 3,4-methylenedioxyamphetamine (MDA), barbiturates, benzodiazepines, cocaine, benzoyl styrene, buprenorphine, cannabinoids, methadone, hydrocodone, ethanol, ethyl glucuronide, ketamine, meperidine, methamphetamine, opioids, oxycodone, morphine, or their metabolites.

23. The ED fragment of claim 22, wherein the drug is MDA.

24. The ED fragment of claim 22, wherein the drug is hydrocodone.

25. The ED fragment of claim 22, wherein the drug is cocaine or benzoyl succinate.

26. The kit according to any one of claims 19-24, wherein the kit comprises a substrate of the β-gal enzyme.

27. A method for detecting a drug in a sample, the method comprising: Combine the following: The ED fragment of any one of claims 1-14, wherein the ED fragment is conjugated with the drug, The enzyme receptor (EA) fragment of β-galactosidase, Antibodies that specifically bind to the drug, The sample, and β-gal enzyme substrate; and Measuring signals that indicate the remodeling of active β-galactase. The presence of a signal indicating the remodeling of active β-galase indicates the presence of a drug in the sample, while the absence of such a signal indicates the absence of a drug in the sample.

28. The method of claim 27, wherein the drug is selected from: fentanyl, heroin, amphetamine, 3,4-methylenedioxyamphetamine (MDA), barbiturates, benzodiazepines, cocaine, benzoyl styrene, buprenorphine, cannabinoids, methadone, hydrocodone, ethanol, ethyl glucuronide, ketamine, meperidine, methamphetamine, opioids, oxycodone, morphine, or metabolites thereof.

29. The method of claim 27, wherein the drug is MDA.

30. The method of claim 27, wherein the drug is hydrocodone.

31. The method of claim 27, wherein the drug is cocaine or benzoyl succinate.

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