Βeta-galactosidase enzyme-based immunoassay

Description

[0001] BETA-GALACTOSIDASE ENZYME-BASED IMMUNOASSAY

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 390,544, filed July 19, 2022, which application is incorporated herein by reference in its entirety.

[0004] INTRODUCTION

[0005] Analysis of a biological sample often involves determining presence of a target analyte in the sample. The target analyte may be present in minute quantities, requiring a sensitive assay for detection of the target analyte. In many applications, in addition to sensitivity, speed and simplicity in obtaining analysis results are desirable.

[0006] Beta-galactosidase (P-gal) enzyme-based complementation assays have been utilized in detection of a target analyte. Such a complementation assay usually measure association of an enzyme donor (ED) fragment and an enzyme acceptor (EA) fragment of p-gal into an enzymatically active p-gal.

[0007] SUMMARY

[0008] An enzyme donor (ED) fragment of a beta-galactosidase (p-gal) enzyme is provided. The ED fragment comprises an amino acid sequence having at least 90% amino acid identity to the following amino acid sequence:

[0009] CPGNIDPCASSNSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTD CPSQQLCQ (SEQ ID NO:1 ), and comprises: i) a cysteine (C) residue at position 1 , 8, 53, and 59; ii) a C at position 8 and an acetylated N-terminus; iii) a C at position 1 , 8, and 53; iv) a C at position 8 and 53 and an acetylated N-terminus; or v) a C at position 53 and an acetylated N-terminus, where the residue position is with reference to the residue position in SEQ ID

[0010] NO:1. In certain aspects, the amino acid sequence of the ED fragment has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:1 . In certain aspects, 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.

[0011] In certain aspects, the amino acid sequence of the ED fragment comprises i) a cysteine (C) residue at position 1 , 8, 53, and 59 and an acetylated N-terminus. In certain aspects, the ED fragment comprises only four C residues.

[0012] In certain aspects, the amino acid sequence of the ED fragment comprises ii) a C at position 8 and an acetylated N-terminus. In certain aspects, the ED fragment comprises only one C residue.

[0013] In certain aspects, the amino acid sequence of the ED fragment comprises iii) a C at position 1 , 8, and 53 and an acetylated N-terminus. In certain aspects, the ED fragment comprises only three C residues.

[0014] In certain aspects, the amino acid sequence of the ED fragment comprises iv) a C at position 8 and 53 and an acetylated N-terminus. In certain aspects, the ED fragment comprises only two C residues.

[0015] In certain aspects, the amino acid sequence of the ED fragment comprises v) a C at position 53 and an acetylated N-terminus. In certain aspects, the ED fragment comprises only one C residue.

[0016] In certain aspects, the C-terminus of the amino acid sequence of the ED fragment comprises an amide modification.

[0017] In certain aspects, the ED is conjugated to a drug and can associate with an enzyme acceptor (EA) fragment of the b-galactosidase to form a catalytically active 0- gal enzyme.

[0018] In certain aspects, the drug is selected from the group consisting of: fentanyl, heroin, amphetamine, 3,4-Methylenedioxyamphetamine (MDA), barbiturate, benzodiazepine, cocaine, Benzoylecgonine, Buprenorphine, Cannabinoid, Methadone, hydrocodone, ethyl alcohol, Ethyl Glucuronide, Ketamine, Meperidine, Methamphetamine, Opiate, Oxycodone, morphine, or a metabolite thereof. In certain aspects, the drug is MDA. In certain aspects, the drug is cocaine or Benzoylecgonine.

[0019] The present disclosure also provides a kit comprising the ED fragment as disclosed herein and an EA fragment, where the ED and EA fragment associate to form an catalytically active p-gal enzyme. The kit may further include an antibody that specifically binds to a drug. In certain aspects, the ED fragment is conjugated to the drug. The drug may be a drug selected from the group consisting of: fentanyl, heroin, amphetamine, 3,4-Methylenedioxyamphetamine (MDA), barbiturate, benzodiazepine, cocaine, Benzoylecgonine, Buprenorphine, Cannabinoid, Methadone, hydrocodone, ethyl alcohol, Ethyl Glucuronide, Ketamine, Meperidine, Methamphetamine, Opiate, Oxycodone, morphine, or a metabolite thereof. The kit may further include a substrate for the p-gal enzyme.

[0020] Also disclosed herein is a method for detecting a drug in a sample. The method may include combining: an ED fragment as disclosed herein, where the ED fragment is conjugated to the drug, an enzyme acceptor (EA) fragment of the p-gal enzyme, an antibody that specifically binds to the drug, the sample, and a p-gal enzyme substrate; and measuring a signal indicative of reconstitution of an active p-gal enzyme, where presence of a signal indicative of reconstitution of the active p-gal enzyme indicates presence of the drug in the sample and where absence of a signal indicative of reconstitution of the active p-gal enzyme indicates absence of the drug in the sample.

[0021] In certain aspects, the drug is selected from the group consisting of: fentanyl, heroin, amphetamine, 3,4-Methylenedioxyamphetamine (MDA), barbiturate, benzodiazepine, cocaine, Benzoylecgonine, Buprenorphine, Cannabinoid, Methadone, hydrocodone, ethyl alcohol, Ethyl Glucuronide, Ketamine, Meperidine, Methamphetamine, Opiate, Oxycodone, morphine, or a metabolite thereof.

[0022] BRIEF DESCRIPTION OF THE FIGURES

[0023] FIG. 1 depicts an exemplary assay for detection of a drug in a sample. In this assay, the sample, suspected of containing the drug is combined with an antibody that binds to the drug, an enzyme acceptor (EA) fragment of p-gal, and an enzyme donor (ED) fragment of p-gal conjugated to the drug (ED-drug). If not drug is present in the sample, the antibody binds to the ED-drug thereby sequestering the ED-drug and preventing association of ED-drug with EA. Upon addition of a substrate for p-gal, no signal is detected, indicating absence of detectable level of drug in the sample. If drug is present in the sample, the antibody binds to the drug, allowing ED-drug to associate with EA fragment to form a catalytically active b-gal enzyme which is detectable upon addition of a b-gal substrate. Present of a signal is indicative of presence of the drug in the sample. FIG. 2 depicts a schematic showing modifications made to an ED fragment. “SH” indicates presence of a cysteine residue. “NH2” indicates amination.

[0024] FIG. 3 shows a plot of signal (Y-axis) indicative of presence of 3,4- Methylenedioxyamphetamine (MDA) at concentrations indicated on the X-axis. MDA was detected using standard ED60-MDA conjugate, ED60-MDA conjugate complexed with additional molecules of MDA, and ED fragment mutant D-MDA.

[0025] FIG. 4 shows a plot of signal (Y-axis) indicative of presence of hydrocodone at concentrations indicated on the X-axis. Hydrocodone was detected using standard ED60-hydrocodone conjugate and ED fragment mutant D-hydrocodone.

[0026] FIG. 5 shows a plot of signal (Y-axis) indicative of presence of Benzoylecgonine at concentrations indicated on the X-axis. Benzoylecgonine was detected using standard ED60-Benzoylecgonine conjugate and ED fragment mutant D-Benzoylecgonine.

[0027] DETAILED DESCRIPTION

[0028] Novel polypeptides that serve as the enzyme donor (ED) fragment of a complex with the enzyme acceptor (EA) fragment of beta-galactosidase are provided. The ED fragment comprises an amino acid sequence having at least 90% amino acid identity to the following amino acid sequence:

[0029] CPGNIDPCASSNSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTD CPSQQLCQ (SEQ ID NO:1 ), and comprises: i) a cysteine (C) residue at position 1 , 8, 53, and 59; ii) a C at position 8 and an acetylated N-terminus; iii) a C at position 1 , 8, and 53; iv) a C at position 8 and 53 and an acetylated N-terminus; or v) a C at position 53 and an acetylated N-terminus, where the residue position is with reference to the residue position in SEQ ID NO:1 .

[0030] Before the present invention is described in greater detail, it is to be understood that the present disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed.

[0031] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating un-recited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0032] 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 belongs.

[0033] The present disclosure may be understood more readily by reference to the following detailed description of desired embodiments and the examples included therein. In the following specification and the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings.

[0034] Although specific terms are used in the following description for the sake of clarity, these terms are intended to refer only to the particular structure of the embodiments selected for illustration in the drawings, and are not intended to define or limit the scope of the disclosure. In the drawings and the following description below, it is to be understood that like numeric designations refer to components of like function.

[0035] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0036] The term “comprising” is used herein as requiring the presence of the named component and allowing the presence of other components. The term “comprising” should be construed to include the term “consisting essentially of” and “consisting of”. The “consisting essentially of” allows the presence of the named component(s), along with other component which do not change the function / structure of the named component(s). The “consisting of” allows the presence of the named component(s), along with any adhesives or other bonding means for attaching the listed component(s).

[0037] Numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0038] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 2 grams to 10 grams” is inclusive of the endpoints, 2 grams and 10 grams, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0039] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context. When used in the context of a range, the modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the range of from about “2 to about 10” also discloses the range “from 2 to 10.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11 %, and “about 1 ” may mean from 0.9-1.1.

[0040] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0041] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation.

[0042] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0043] BETA-GAL ENZYME DONOR FRAGMENT

[0044] As summarized above, aspects of the present disclosure include an ED fragment that includes an amino acid sequence having at least 90% amino acid identity to the following amino acid sequence:

[0045] CPGNIDPCASSNSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTD CPSQQLCQ (SEQ ID NO:1 ), and comprises: i) a cysteine (C) residue at position 1 , 8, 53, and 59; ii) a C at position 8 and an acetylated N-terminus; iii) a C at position 1 , 8, and 53; iv) a C at position 8 and 53 and an acetylated N-terminus; or v) a C at position 53 and an acetylated N-terminus, where the residue position is with reference to the residue position in SEQ ID NO:1 .

[0046] In certain aspects, the amino acid sequence of the ED fragment has at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:1. In certain aspects, the amino acid sequence of the ED fragment includes conservative substitutions relative to the amino acid sequence of SEQ ID NO:1. For the most part, conservative substitutions are involved, where the non-polar aliphatic amino acids, such as G, A, V, L, and I may be substituted one for the other, the non-charged polar amino acids, such as C, M, S, T, N, and Q may be substituted one for the other, the charged amino acids may be substituted one for the other of the same charge, i.e. K and R; and D and E; and the aromatic amino acids may be substituted one for the other, F, W, and Y.

[0047] In certain aspects, 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 certain aspects, 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 certain aspects, the amino acid sequence of the ED fragment may be increased by up to up to 5, 10, 20, 30, 40, 50, or 60 amino acids relative to SEQ ID NO:1 , where the added amino acids are the same as those present in the corresponding position in a naturally occurring 0-gal enzyme.

[0048] In certain aspects, the amino acid sequence of the ED fragment comprises i) a cysteine (C) residue at position 1 , 8, 53, and 59 and an acetylated N-terminus. In certain aspects, the ED fragment comprises only four C residues. In certain aspects, the amino acid sequence of the ED fragment is as set forth in SEQ ID NO:1 . In certain aspects, the ED fragment is acetylated at the N-terminus and includes an amide at the C-terminus.

[0049] In certain aspects, the amino acid sequence of the ED fragment comprises ii) a C at position 8 and an acetylated N-terminus. In certain aspects, the ED fragment comprises only one C residue. In certain aspects, the amino acid sequence of the ED fragment comprises the amino acid sequence set forth in SEQ ID NO: 5. In certain aspects, the ED fragment is acetylated at the N-terminus and includes an amide at the C-terminus.

[0050] In certain aspects, the amino acid sequence of the ED fragment comprises iii) a C at position 1 , 8, and 53 and an acetylated N-terminus. In certain aspects, the ED fragment comprises only three C residues. In certain aspects, the amino acid sequence of the ED fragment comprises the amino acid sequence set forth in SEQ ID NO: 6. In certain aspects, the ED fragment is acetylated at the N-terminus and includes an amide at the C-terminus.

[0051] In certain aspects, the amino acid sequence of the ED fragment comprises iv) a C at position 8 and 53 and an acetylated N-terminus. In certain aspects, the ED fragment comprises only two C residues. In certain aspects, the amino acid sequence of the ED fragment comprises the amino acid sequence set forth in SEQ ID NO: 7. In certain aspects, the ED fragment is acetylated at the N-terminus and includes an amide at the C-terminus.

[0052] In certain aspects, the amino acid sequence of the ED fragment comprises v) a C at position 53 and an acetylated N-terminus. In certain aspects, the ED fragment comprises only one C residue. In certain aspects, the amino acid sequence of the ED fragment comprises the amino acid sequence set forth in SEQ ID NO: 8. In certain aspects, the ED fragment is acetylated at the N-terminus and includes an amide at the C-terminus. As used herein, “Amino” refers to the — NH2, “Carboxyl” refers to the — CO2H, “Acetyl” refers to the — C(O)CHs Protein Nt-acetylation refers to the covalent attachment of an acetyl group (CH3CO) to the free a-amino group (NHs+) at the N- terminal end of a polypeptide. C-terminal amidation removes the charge from the C- terminus of a peptide. C-terminal amidation of the ED fragment disclosed herein may include attachment of amide (NH2) or N-alkyl amide to the C-terminus. “Alkyl” refers to a saturated, straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, having from one to twelve carbon atoms (C1-C12 alkyl), from one to eight carbon atoms (Ci-Cs alkyl) or from one to six carbon atoms (C1-C6 alkyl), and which is attached to the rest of the molecule by a single bond.

[0053] The ED may be joined to any compound of interest. The EDs of the subject invention can be used in competitive and non-competitive assays for detecting the presence of drugs, polypeptides (e.g., antibodies), lectins, nucleic acids, sugars, or other analyte of interest. For examples of the use of EDs for the determination of analytes, see for example, U.S. Pat. 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, which are specifically incorporated by reference as illustrating the manner of performing the assays, where the EDs of the subject invention may be directly substituted for the EDs employed in the indicated references.

[0054] The ED may be prepared by any convenient means. Where the ED is joined to other than a polypeptide, it may be synthesized by conventional means using commercially available synthesizers or it may be prepared using cloning, where the ED may be produced intracellularly and isolated by lysing or may be secreted, using an appropriate signal sequence. However, where a fusion protein is employed and the fusion protein substantially exceeds about 60 amino acids, usually cloning will be employed, where the protein may be isolated by lysis or from the medium by secretion as described above.

[0055] ED fragments may be synthesized on an automated peptide synthesizer by standard synthetic techniques. In certain aspects, a protected amino acid representing the carboxy terminus amino acid of the desired peptide is attached to cross-linked polystyrene beads. The resin beads function as a solid phase to which additional amino acids may be coupled in a step-wise manner. The peptide is generated by growing the chain sequentially from the carboxy-terminus to the N-terminus. The solid phase facilitates driving the reaction rapidly to 100% completion by the use of excess reagents. The excess reagents can then be easily washed away. Upon completion of the synthetic steps, the peptide is removed from the resin and purified.

[0056] ED-Druq Conjugates

[0057] In certain aspects, the ED is conjugated to a drug and can associate with an enzyme acceptor (EA) fragment of the p-galactosidase to form a catalytically active 0- gal enzyme.

[0058] In certain aspects, the drug is selected from the group consisting of: fentanyl, heroin, amphetamine, 3,4-Methylenedioxyamphetamine (MDA), barbiturate, Secobarbital, Diazepam, Oxazepam, benzodiazepine, cocaine, Benzoylecgonine, Buprenorphine, Cannabinoid, Methadone, hydrocodone, ethyl alcohol, Ethyl Glucuronide, Ketamine, Meperidine, Methamphetamine, Opiate, Oxycodone, morphine, Quinidine, Propoxyphen, N-acetylprocainamide, Tobramycin or a derivative (e.g., a metabolite) thereof, Estriol, Digoxin, Thyroxine, Propranolol , Methotrexate , phencyclidine, Methadone , Gentamicin, Theophylline, Benzoyl ecogonine, Phenytoin, Procainamide, Lidocaine, Carbamazepine, Primidene, Valproic Acid, Phenobarbital, Ethosuxinimide, Biotin or a metabolite or derivative thereof. In certain aspects, the drug is MDA. In certain aspects, the drug is cocaine or Benzoylecgonine.

[0059] The ED fragments disclosed herein include amino acids, such as, cysteine or lysine introduced for coupling of analytes to the ED fragments. When a free sulfhydryl is present, it can react with a reactive group that is present on the analyte. Such reactive groups include but are not limited to, reactive haloalkyl groups and acid / halo groups, p- mecuribenzoate groups and groups capable of Michael-type addition reactions (including, for example, maleimides and groups of the type described in Mitral and Lawton, 1979, J. Amer. Chem. Soc. 101 :3097-3110). Haloalkyl as defined herein comprises any alkyl group from one to three carbon atoms substituted with bromine, iodine or chlorine. If the analyte does not possess such reactive group for coupling to the free sulfhydryl of the enzyme-donor, a derivative of the analyte can be prepared to contain such reactive group.

[0060] ED-Polypeptide Fusions

[0061] In certain aspects, the ED fragment may be conjugated to a heterologous polypeptide. As used herein, the term “heterologous” refers to a component of a polypeptide that is non-native to the polypeptide, i.e., not typically found in nature in association with the rest of the polypeptide to which it is being compared.

[0062] According to another embodiment of the present invention, an enzyme-donor polypeptide is prepared by ligating or fusing a gene encoding ED with another gene encoding a heterologous protein analyte (or a portion thereof) to be assayed. The expression of the ligated genes in an appropriate host cell results in a fusion protein product that is capable both of complementation with an enzyme-acceptor and specific binding to an analyte-binding protein, e.g., a receptor or an antibody. Thus, fusion proteins prepared according to this embodiment of the present invention comprise two domains: (1 ) ED domain, and (2) a protein domain, both encoded by a fused gene. A spacer sequence between the ED and the protein domain can be used to enhance complementation.

[0063] The analyte may be human glycoproteins leutropin, leutinizing hormone, follitropin, follicle-stimulating hormone, thyrotropin, thyroid-stimulating hormone or human chorionic gonadotropin (hCG) Carcinoembryonic antigen, Ferritin, Human T-Cell Leukemia Virus, Insulin, o-fetoprotein, Herpesvirus, Cytomegalovirus, Follicle stimulating hormone, thyroid stimulating hormone, Luteinizing hormone, hepatitis Virus, chorionic Gonadotropin, Estrogen receptor , thyroid stimulating hormone receptor, Poliovirus receptor, Insulin transport protein, Protein A, Con A lectin, Wheat Germ agglutinin lectin, Secretory protein, Cholera toxin, or Avidin.

[0064] The ED-analyte fusion may be used in conjunction with an EA and a polypeptide (e.g., an antibody or a receptor) that binds to the analyte to detect presence of the analyte in a biological sample.

[0065] EA FRAGMENT

[0066] P-galactosidase is a tetrameric protein having a molecular weight (MW) equal to 540,000 daltons. The four identical monomers consist of 1021 amino acids, each with a MW of 1 16,000 daltons. The monomeric protein is divided into three regions: (1 ) the N- terminal proximal segment (the a-region), (2) a middle region, and (3) a C-terminal distal segment (the co-region).

[0067] In certain aspects, EA that may be used in the methods and kits disclosed herein is has a sequence of the beta-galactosidase Enzyme Acceptor (also known as Omega Domain) which is the larger fragment of the Beta-galactosidase enzyme. This fragment can associate with the ED fragments disclosed herein and reconstitute an active Betagalactosidase enzyme. THE EA fragment may be obtained from a commercial supplier.

[0068] SUBSTRATES

[0069] In certain aspects, the ED is used with the EA to form an active p-galactosidase enzyme that can be detected by the addition of a detectable substrate, normally a colored, fluorescent or chemiluminescent substrate, p-galactosidase effectively uses fluoresces having phenolic groups that are etherified with a p-galactosyl group. The common substrates are p-D-galactopyranosyl phenols, such as fluorescein, mono- and di-susbtituted, o-nitrophenyl-p-D-galactoside, p-methylumbelliferyl-p-D-galactoside, X- gal, resorufin-p-D-galactoside, commercially available oxetanes, e.g., Galacto-Light Plus® kits (chemiluminescence) and chlorophenol red. Substrates for p-galactosidase that are of particular interest, especially due to their suitablity for spectrophotometric or fluorometric analysis include, but are not limited to: p-aminophenyl-p-D- galactopyranoside; 2'-N-(hexadecanol)-N-(amino-4'-nitrophenyl)-p-D-galactopyranoside; 4-methylumbelliferyl-p-D-galactopyranoside; napthyl-AS-BI-p-D-galactopyranoside; 1- napthyl-p-D-galactopyranoside; 2-napthyl-p-D-galactopyranoside monohydrate; 0- nitrophenyl-p-D-galactopyranoside; m-nitrophenyl-p-D-galactopyranoside; p-nitrophenyl- p-D-galactopyranoside; and phenyl-p-D-galactopyranoside, 5-bromo-4-chloro-3-indolyl-p- D-galactopynanoside, resorufin-p-D-galactopyranoside, 7-hydroxy-4-trifluoromethyl coumarin, uj-nitrostyryl-p-D-galactopyranoside, and flourescein-p-D-galactopyranoside.

[0070] ENZYME COMPLEMENTATION ASSAY

[0071] The amount of unknown analyte in a sample mixture can be measured as a direct function of the activity of the p-galactosidase enzyme. Enzyme activity is monitored by the appearance of a product of the enzymatically catalyzed reaction or by disappearance of the enzyme substrate. This is the rate of conversion of substrate.

[0072] Advantageously, the assays disclosed herein are homogenous assays not requiring separation of unbound components, unconverted substrate, purification of reaction product and the like. Such as assay easily lends to automation. Enzyme activity is measured by monitoring the rate of substrate conversion by the enzyme catalyzed reaction by any of a variety of techniques including but not limited to spectrophotometric, and fluorometric methods.

[0073] In certain aspects, the enzyme complementation assay is an immunoassay. The immunoassay may involve the ED-analyte conjugate, EA, an analyte-binding molecule (e.g., an antibody, such as, a monoclonal or polyclonal antibody), and a beta-gal substrate. If the analyte is present in the sample, the analyte competes with the ED- analyte conjugate to bind to the analyte-binding molecule, leading to an increase in the amount of ED-analyte conjugate available to associate with the EA to form an enzymatically active beta-gal.

[0074] In certain aspects, the methods for detecting presence and optionally concentration of an analyte in a sample may involve combining an ED fragment as disclosed herein, where the ED fragment is conjugated to the analyte, an enzyme acceptor (EA) fragment of the beta-gal enzyme, a polypeptide (e.g., an antibody) that specifically binds to the analyte, the sample, and a beta-gal enzyme substrate. The combining may be performed in a single step or any number of the individual components may be pre-mixed prior to combining with the sample and / or the susbtrate. The combined mixture may be optionally incubated for any suitable period of time at room temperature, at 4oC, 30oC, 37oC, or another suitable temperature.

[0075] The method may further include measuring a signal indicative of reconstitution of an active beta-gal enzyme, where presence of a signal indicative of reconstitution of the active beta-gal enzyme indicates presence of the drug in the sample and wherein absence of a signal indicative of reconstitution of the active beta-gal enzyme indicates absence of the drug in the sample.

[0076] ANALYTES

[0077] The EDs of the present invention can be used to determine the presence and / or quantity of a variety of analytes including drugs and drug metabolites, biologically active molecules, steroids, vitamins, industrial pollutants, pesticides and their metabolites, food additives, herbicides and their metabolites, flavoring agents and food poisons, pathogens and toxins they produce, and other substances of interest. Analytes of relatively high molecular weight, e.g., proteins with MW greater than about 2,000 daltons, as well as smaller analytes can be detected and / or measured with the disclosed EDs. Illustrative examples of such analytes include, but are not limited to, the following drugs fentanyl, heroin, amphetamine, 3,4-Methylenedioxyamphetamine (MDA), barbiturate, Secobarbital, Diazepam, Oxazepam, benzodiazepine, cocaine, Benzoylecgonine, Buprenorphine, Cannabinoid, Methadone, hydrocodone, ethyl alcohol, Ethyl Glucuronide, Ketamine, Meperidine, Methamphetamine, Opiate, Oxycodone, morphine, Quinidine, Propoxyphen, N-acetylprocainamide, Tobramycin or a derivative (e.g., a metabolite) thereof, Estriol, Digoxin, Thyroxine, Propranolol , Methotrexate , phencyclidine, Methadone , Gentamicin, Theophylline, Benzoyl ecogonine, Phenytoin, Procainamide, Lidocaine, Carbamazepine, Primidene, Valproic Acid, Phenobarbital, Ethosuxinimide, Biotin or a metabolite or derivative thereof.

[0078] In certain aspects, the analyte is selected from Carcinoembryonic antigen, Ferritin, Human T-Cell Leukemia Virus, Insulin, o-fetoprotein, Herpesvirus, Cytomegalovirus, Follicle stimulating hormone, thyroid stimulating hormone, Luteinizing hormone, hepatitis Virus, chorionic Gonadotropin, Estrogen receptor , thyroid stimulating hormone receptor, Poliovirus receptor, Insulin transport protein, Protein A, Con A lectin, Wheat Germ agglutinin lectin, Secretory protein, Cholera toxin, or Avidin.

[0079] KITS

[0080] The components of the enzyme complementation assays of the present invention may be packaged in a kit either in aqueous medium or in lyophilized form. Each component or reagent can be packaged either separately or with another component so long as the sensitivity of the assay is not altered and the component is not adversely affected. The aqueous medium may include a buffer, a protease inhibitor, and / or a preservative. In certain aspects, the substrate may be present in an opaque container to protect it from light.

[0081] The kits may optionally include a positive control and / or a negative control. In certain aspects, the kit may include the analyte at a plurality of known concentrations for use in calibrating an instrument for detecting a signal produced during assay for the analyte and / or generating a concentration curve for the analyte. EXAMPLES

[0082] BETA-GALACTOSIDASE ENZYME-BASED IMMUNOASSAY

[0083] The following enzyme donor fragments were generated and compared to AIA

[0084] ED60. AIA ED60 contains two C residues. Table 1 shows the results: FIG. 1 shows a schematic of b-galactosidase based immunoassay for detection of drug in a sample. FIG. 2 shows a schematic of ED fragments tested. ED60-D performed the best.

[0085] ED60-D was further modified as listed in Table 2:

[0086] EDs were conjugated to drugs as described below and assayed as per the schematic shown in FIG. 1 .

[0087] Synthesis of MDA derivative:

[0088] Procedure: MDA HOI salt (100mg) was dissolved in 5% sodium bicarbonate (20ml), extracted with ethyl acetate (3x20ml), combined and washed with water (3x30ml), dried with sodium sulfate, filtered and evaporated in vacuo to give 50mg of MDA. The MDA was dissolved in DMF (2ml)at room temperature, to the solution were added bromoacetyl glycine NHS ester (98mg, 1.2eq) and DIPEA (15pl)respectively. Stirred at room temperature for 2 hours, diluted with ethyl acetate (100ml); washed with 5% sodium bicarbonate solution (20ml); followed by water (3x30ml); dried with sodium

[0089] 16

[0090] SUBSTITUTE SHEET ( RULE 26) sulfate; filtered, evaporated in vacuo to give the product (75mg). MS: 357(M+H+, 100%), 359(M+2+H+, 98%).

[0091] Synthesis of BE or Cocaine derivative:

[0092] Procedure: Benzoylecgonine (50mg) was dissolved in DMF (1 ml) at room temperature; to the solution were added tributylamine (48pl), and IBCF (22pl, 1.2eq); stirred at r.t. for 1 hour; N-(2-aminoethyl)maleimide TFA salt (48mg) was added to the solution; stirred at r.t. overnight; 1 ml water was added, the solvents were remove under reduced pressure; loaded onto a prepacked column; purified with an ISCO column (MeOH / CH2Cl2 (0.1% EtsN) to give 50mg product. MS: 412 (M+H+).

[0093] Synthesis of Hydrocodone derivative: Hydromorphone HCI salt (200mg) was suspended in benzene (3ml), to the suspension were added ethylene glycol (2ml) and TsOH (300mg), heated and kept refluxing for 30 minutes, cooled to r.t.; 30ml 5% sodium bicarbonate was added to solution; extracted with ethyl acetate (3x20ml); washed with water (3x30ml); dried with

[0094] 17

[0095] SUBSTITUTE SHEET ( RULE 26) sodium sulfate, filtered and evaporated in vacuo to afford 6-ehylene acetalhydromorphone (white solid, 160mg). MS: 330 (M+H+)

[0096] 6-ehylene acetal- hydromorphone (160mg), PPh3(0.5g) and N-Boc- ethanolamine (0.3g) were dissolved in anhydrous THF (20ml) at r.t. under nitrogen protection; cooled to OoC with an ice-water bath. DIAD (0.4g) was added slowly; kept stirring at r.t. overnight; solvents were removed under reduced pressure; purified with an ISCO column (EtOAc / MeOH 01 % EtsN) to give 160mg of 3-O-Boc-aminoethyl-6- ethylene acetal-hydromorphone. MS: 473 (M+H+).

[0097] 3-O-Boc-aminoethyl-6-ethylene acetal-hydromorphone (160mg) was dissolved in MeOH (6ml); to the solution was added 3N HCI (6ml); heated and kept refluxing for 4 hours. All solvents were removed in vacuo; neutralized with 5% sodium bicarbonate solution and pH was adjusted to 8. The solvent was removed in vacuo; dissolved in acetonitrile (3x30ml); filtered and evaporated in vacuo to afford 3-O-aminoethyl- hydromorphone (100mg). MS: 329(M+H+).

[0098] 3-O-aminoethyl-hydromorphone (1 OOmg) was dissolved in 1 M sodium bicarbonate solution (5ml); cooled to 0°C with an ice water bath, N- (methoxycarbonyl)maleimide (37mg) was added to the solution; stirred at that temperature for 30 minutes; 5ml of acetonitrile was added, stirred at r.t. for 30mintues. Extra 20ml of acetonitrile was added the turbid solution; stirred and filtered; rinsed with ACN (5ml); pH was adjusted to 5 with 6N HCI solution; solvents were removed in vacuo; dissolved in ethanol (10ml); filtered, evaporated in vacuo to afford hydromorphone-3-maleimide (100mg). MS: 409 (M+H+).

[0099] Beta-galactosidase donor peptide mutant D bioconjugation with MDA-Br MDA-Br derivative HPLC purification:

[0100] 1 . 1 mg MDA-Br derivative was dissolved in the mixture of 0.5ml HPLC buffer B (0.1 %TFA acetonitrile) and 0.7ml HPLC buffer A (0.1 % TFA water).

[0101] 2. 1 ,2ml or 1 mg of the derivative solution was injected into HPLC. Waters HPLC ( PDA996, 600 controller, binary pump).. , Phenomenex Kinetex® 5 pm XB C-18

[0102] 10oAcolumn (4.6*250mm). HPLC method: linear gradient: started with 10% acetonitrile, 40% acetonitrile at 30 minutes, followed by 100% acetonitrile. 1 ml / min flow rate Peak at 25.8 min elution time is the purified MDA-Br with correct Mass Spectrometry analysis.

[0103] 3. Structure of MDA-Br derivative

[0104]

[0105] Peptide mutant D HPLC purification:

[0106] 1 . 1 mg of peptide mutant D was dissolved in 1 ml HPLC buffer A ( 0.1 % TFA water)

[0107] 2. 0.5ml or 0.5mg of the peptide mutant solution was injected into HPLC. Waters HPLC( PDA996, 600 controller, binary pump). Phenomenex Kinetex® 5 pm XB C-18 100A column (4.6*250mm), HPLC method: linear gradient: started with 5% acetonitrile, 45% acetonitrile at 35 minutes, followed by 100% acetonitrile.1 ml / min flow rate. Peak at 34.7 min elution time is the purified mutant D and will be used in next step conjugation.

[0108] MDA-Br mutant peptide D conjugate preparation:

[0109] 1 . 212 pl or 0.5mg of the peptide mutant HPLC fraction with 34.7 min elution time was mixed with 510 pl or 1 mg of the MDA-Br HPLC fraction with 25.8 min elution time.

[0110] 2. Adjust the mixture’s pH between 7.0 and 7.5 using 10Oul 0.1 M Phosphate buffer pH 8.0 and suitable amount of 6N NaOH.

[0111] 3. Room temperature rotate for 4 hours.

[0112] 4. Conjugate HPLC purification: Dissolve 400pl of the coupling mixture with 1 ml HPLC buffer A, which was injected into HPLC. Waters HPLC( PDA996, 600 controller, binary pump). Phenomenex Kinetex® 5 pm XB C-18 100A column (4.6*250mm). HPLC method: linear gradient: started with 10% acetonitrile, 45% acetonitrile at 30 minutes, followed by 100% acetonitrile.1 ml / min flow rate. MDA-Br peptide mutant D conjugate is the peak with 32.1 min elution time with correct immunoassay performance.

[0113] FIGS. 3-5 show detection of various drugs using ED60.

[0114] Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

Claims

CLAIMSWHAT IS CLAIMED IS:1 . An enzyme donor (ED) fragment of p-galactosidase (p-gal) enzyme comprising an amino acid sequence having at least 90% amino acid identity to the following amino acid sequence:CPGNIDPCASSNSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTD CPSQQLCQ (SEQ ID NO:1), and comprising: i) a cysteine (C) residue at position 1 , 8, 53, and 59; ii) a C at position 8 and an acetylated N-terminus;Hi) a C at position 1 , 8, and 53; iv) a C at position 8 and 53 and an acetylated N-terminus; or v) a C at position 53 and an 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 to 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) a cysteine (C) residue at position 1 , 8, 53, and 59 and an acetylated N-terminus.

5. The ED fragment of claim 4, wherein the ED fragment comprises 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 comprises only oneC residue.

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

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

10. The ED fragment of claim 1 , wherein the amino acid sequence comprises iv) a C at position 8 and 53 and an acetylated N-terminus.11 . The ED fragment of claim 10, wherein the ED fragment comprises 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 comprises only one C residue.

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

15. The ED fragment of any one of claims 1-14, wherein the ED is conjugated to a drug and can associate with an enzyme acceptor (EA) fragment of the □- galactosidase to form a catalytically active f3-gal enzyme.

16. The ED fragment of claim 15, wherein the drug is selected from the group consisting of: fentanyl, heroin, amphetamine, 3, 4-Methylenedioxyamphetamine (MDA), barbiturate, benzodiazepine, cocaine, Benzoylecgonine, Buprenorphine, Cannabinoid, Methadone, hydrocodone, ethyl alcohol, Ethyl Glucuronide, Ketamine, Meperidine, Methamphetamine, Opiate, Oxycodone, morphine, or a metabolite 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 Benzoylecgonine.

19. A kit comprising: the ED fragment of any one of claims 1 -14; and an enzyme acceptor (EA) fragment of the 0-gal enzyme.

20. The kit of claim 19, further comprising an antibody that specifically binds to a drug.21 . The kit of claim 20, wherein the ED fragment is conjugated to the drug.

22. The kit of claim 20, wherein the drug is selected from the group consisting of: fentanyl, heroin, amphetamine, 3, 4-Methylenedioxyamphetamine (MDA), barbiturate, benzodiazepine, cocaine, Benzoylecgonine, Buprenorphine, Cannabinoid, Methadone, hydrocodone, ethyl alcohol, Ethyl Glucuronide, Ketamine, Meperidine, Methamphetamine, Opiate, Oxycodone, morphine, or a metabolite thereof.

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 orBenzoylecgonine.

26. The kit of any one of claims 19-24, wherein kit comprises a substrate for the p-gal enzyme.

27. A method for detecting a drug in a sample, the method comprising: combining:an ED fragment of any one of claims 1-14, wherein the ED fragment is conjugated to the drug, an enzyme acceptor (EA) fragment of the P-gal enzyme, an antibody that specifically binds to the drug, the sample, and a p-gal enzyme substrate; and measuring a signal indicative of reconstitution of an active p-gal enzyme, wherein presence of a signal indicative of reconstitution of the active p-gal enzyme indicates presence of the drug in the sample and wherein absence of a signal indicative of reconstitution of the active p-gal enzyme indicates absence of the drug in the sample.

28. The method of claim 27, wherein the drug is selected from the group consisting of: fentanyl, heroin, amphetamine, 3, 4-Methylenedioxyamphetamine (MDA), barbiturate, benzodiazepine, cocaine, Benzoylecgonine, Buprenorphine, Cannabinoid, Methadone, hydrocodone, ethyl alcohol, Ethyl Glucuronide, Ketamine, Meperidine, Methamphetamine, Opiate, Oxycodone, morphine, or a metabolite 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 Benzoylecgonine.

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