PREPARATION OF MULTI-HAPTEN MUTANT G6PDH CONJUGATES AND THEIR USE FOR DETECTION OF MULTIPLE ANALYTES

Abstract

The present invention relates to multi-hapten mutant G6PDH conjugates, methods of their preparation and their use for detection of multiple analytes. The compositions of the invention comprise different types of haptens (molecules) require to be immobilized into the G6PDH to make one multi-hapten-G6PDH conjugate. Both thiol and amine functional groups on G6PDH are utilized for immobilization of different haptens.

Claims

1-11. (canceled)

12. A method for producing a composition, the composition comprising at least two distinct specific binding pair (sbp) members conjugated to a mutated glucose-6-phosphate dehydrogenase (G6PDH), wherein the mutation comprises the insertion or substitution of at least one cysteine amino acid residue and wherein at least a first specific binding pair member is conjugated to said mutated G6PDH via a thiol group on the cysteine and wherein at least a further specific binding pair member is conjugated to G6PDH via an amino group, the method comprising the steps of: conjugating a first specific binding pair member to mutant G6PDH via a thiol group; and then conjugating at least a further specific binding pair member to mutant G6PDH via an amino group.

13. The method according to claim 12, wherein the step of conjugating includes providing a linker between G6PDH and either or both of specific binding pair member and further specific binding pair member.

14. (canceled)

15. The method of claim 12, wherein mutant G6PDH is a bacterial G6PDH derived from the genus of Leuconostoc.

16. The method of claim 12, wherein the two distinct binding pair members are serologically not cross-reactive.

17. The method of claim 12, wherein the composition comprises three or more distinct specific binding pair (sbp) members conjugated to G6PDH.

18. The method of claim 12, wherein the composition comprises a plurality of distinct further specific binding pair members conjugated to a respective plurality of amino groups.

19. The method of claim 12, wherein the composition comprises a plurality of distinct first specific binding pair members conjugated to a respective plurality of thiol groups.

20. The method of claim 12, wherein the first and second specific binding pair members are selected from the group consisting of opium, opioid analgesics, amphetamines, cocaine, methadone, alkaloids, catecholamines, methylendioxyamphetamines (MDMA, MDA, and MDEA, etc.), PCP, propoxyphene, methaqualone, barbiturates, benzodiazepines, tricyclic antidepressants, tranquilizers, tetrahydrocannabinol, LSD, ketamine, GHB, and other drugs of abuse, including amino acids, hormones, and steroids, buprenorphine, norbuprenorphine, and analogs, metabolites, and derivatives thereof.

21. The method of claim 17, wherein the first binding pair member is MDA or an analog, metabolites, or derivative thereof and the second binding pair member is Amphetamine or an analog, metabolite, or derivative thereof and a third binding pair member is Methamphetamine or an analog, metabolites, or derivative thereof.

Description

[0054] In the attached Figures,

[0055] FIG. 1 schematically shows the chemical structure of embodiments of compositions according to the invention,

[0056] FIG. 2 shows the structure of the exemplary analytes MDMA, MDA and MDEA,

[0057] FIG. 3 shows an exemplary synthesis scheme for obtaining an embodiment of a composition according to the invention, showing the synthesis of MDA/amphetamine mutant G6PDH conjugate (7),

[0058] FIG. 4 shows an exemplary synthesis scheme for obtaining an embodiment of a composition according to the invention, showing the synthesis of MDA/Methamphetamine mutant G6PDH conjugate (9)

[0059] FIG. 5 shows an exemplary synthesis scheme for obtaining an embodiment of a composition according to the invention, showing the synthesis of MDA/Amphetamine/Methamphetamine mutant G6PDH conjugate(10),

[0060] FIG. 6 shows a response curve for detection of ecstasy, MDA, MDEA, methamphetamine and amphetamine using a composition according to the invention,

[0061] FIG. 7 shows a response curve for detection of ecstasy, MDA, MDEA, methamphetamine and amphetamine using a composition according to the invention,

[0062] FIG. 8 shows a response curve for detection of ecstasy, MDA, MDEA, methamphetamine and amphetamine using a composition according to the invention,

[0063] Homogeneous enzyme immunoassays depend on the availability of enzyme-sbp member conjugates whose enzyme activity can be strongly modulated on binding of the sbp partner. The present invention provides enzyme-sbp member conjugates which can bind to and detect multiple different analytes for conducting assays that are useful in homogeneous immunoassays.

[0064] The compositions of the invention with multiple detection capability in EMITformat need to satisfy some basic conditions: 1) different types of haptens (molecules) require to be immobilized into the G6PDH to make one multi-hapten-G6PDH conjugate; 2) the multi-hapten-G6PDH conjugate needs to be recognized by its corresponding antibody(ies) in order to generate enzymatic inhibition and therefore detection of multiple drugs; 3) the multi-hapten-G6PDH conjugate should retain its enzymatic activity. Based on these conditions, mutant G6PDH was selected as an excellent template to make the new multi-hapten-G6PDH conjugate. Both thiol and amine functional groups on G6PDH are utilized for immobilization of different haptens.

[0065] A suitable mutant G6PDH for example is G6PDH from Leuconostoc mesenteroides (e.g. ATCC 12291) carrying one or several of the following mutations:

Ala-45-Cys

Arg-46-Cys

Gln-47-Cys

Ala-48-Cys

Leu-49-Cys

Asn-50-Cys

Asp-51-Cys

Asp-52-Cys

Glu-53-Cys

Phe-54-Cys

Lys-55-Cys

Gln-56-Cys

Leu-57-Cys

Val-58-Cys

Arg-59-Cys

Asp-60-Cys

Lys-128-Cys

Lys-182-Cys

[0066] The entire amino acid sequence of the wild type G6PDH from Leuconostoc mesenteroides is given below and in references 12, 13, and 14:

TABLE-US-00001 VSEIKTLVTFFGGTGDLAKRKLYPSVFNLYKKGYLQKHFA IVGTARQALNDDEFKQLVRDSIKDFTDDQAQAEAFIEHFS YRAHDVTDAASYAVLKEAIEEAADKFDIDGNRIFYMSVAP RFFGTIAKYLKSEGLLADTGYNRLMIEKPFGTSYDTAAEL QNDLENAFDDNQLFRIDHYLGKEMVQNIAALRFGNPIFDA AWNKDYIKNVQVTLSEVLGVEERAGYYDTAGALLDMIQNH TMQIVGWLAMEKPESFTDKDIRAAKNAAFNALKIYDEAEV NKYFVRAQYGAGDSADFKPYLEELDVPADSKNNTFIAGEL QFDLPRWEGVPFYVRSGKRLAAKQTRVDIVFKAGTFNFGS EQEAQEAVLSIIIDPKGAIELKLNAKSVEDAFNTRTIDLG WTVSDEDKKNTPEPYERMIHDTMNGDGSNFADWNGVSIAW KFVDAISAVYTADKAPLETYKSGSMGPEASDKLLAANGDAWVFKG

[0067] In analogy, further suitable mutant forms of G6PDH are derived from other strains of Leuconostoc mesenteroides, from strains of Leuconostoc citreum (e.g. strain NCIMB 3351), Leuconostoc lactis (e.g. strain NCDO 546), and Leuconostoc dextranicum (e.g. strain ATCC 19255). Suitable mutations include the insertion of at least one cysteine per subunit, or substitution of at least one amino acid with cysteine per subunit, as compared to the precursor G6PDH. In particular, suitable mutant forms of G6PDH include mutations wherein a respective amino acid in a precursor G6PDH molecule derived from Leuconostoc is substituted by cystein at any of the positions 45 to 60.

[0068] The reagent formulation is a two-step reaction. First, two haptens are attached to the G6PDH (Schemes 1-3) using the thiol chemistry in a controlled manner. Second, different types of haptens can be immobilized using the amine chemistry. Our approaches are different from that of the existing multi-hapten conjugate (reference 11) in which only amine chemistry was used. Based on existing data, using the amine functional groups for immobilization of haptens could lead to reduction of the enzymatic activity. This means that only limited amine groups can be used for the attachment of haptens in order to maintain good enzymatic activity for the EMITformat. Consequently, using amine chemistry only for preparation of multi-hapten-G6PDH conjugate could limit the amount of hapten on the conjugate in order to maintain good enzymatic activity.

[0069] In our approaches, multi-hapten-mutant G6PDH was prepared using the thiol function groups on the enzyme as the first step reaction. The resulting conjugate retains most of its enzymatic activity 95%). This could be an advantage in that more haptens can be attached to the amine groups in the second step reaction, while still maintaining good enzymatic activity. Therefore, applying both thiol and amine chemistry in preparation of multi-hapten-G6PDH should result in a conjugate that has more hapten loading capacity for the detection of multiple analytes. Without wishing to be bound by this theory, the beneficial effect of utilizing thiol chemistry in thee compositions and methods of the invention could be explained as follows: Utilization of thiol chemistry to could result in more stable. Cystein residues can be used that are distanced from the active site of the enzyme e. In that regard, hapten conjugation through linkage of cysteine may have little or no impact on the stability of the active quaternary structure that is crucial for the enzymatic activity. This is supported by the observation that mutant G6PDH-hapten conjugates retain very good enzymatic.

[0070] There are many analytes, e.g. drugs, that can be used to prepare multi-hapten-G6PDH and test its performance. Three widely abused drugs, amphetamine, methamphetamine and ecstasy were selected as exemplary analytes. With this in mind, multi-hapten-G6PDH (7, 9, 10) was prepared as described in Schemes 1-3 and the experimental section. Two multi-hapten-G6PDHs (7, 9) were used to investigate their multi-drug detection capability in the EMIT format. Results were discussed in the following section.

[0071] In these approaches, multi-hapten-mutant G6PDH was prepared using the thiol function groups on the enzyme as the first step reaction. The resulting conjugate retains most of its enzymatic activity 95%). This could be an advantage in that more haptens can be attached to the amine groups in the second step reaction, while still maintaining good enzymatic activity. Therefore, applying both thiol and amine chemistry in preparation of multi-hapten-G6PDH should result in a conjugate that have more hapten loading capacity for the detection of multiple analytes.

[0072] EMITAssay Principle:

[0073] The Emit II Plus Assay is a homogeneous enzyme immunoassay. It is based on competition between drug in the sample and drug-labeled with glucose-6-phosphate dehydrogenase (G6PDH) for antibody binding sites. The enzyme conjugate activity decreases upon binding to the antibody. The unbound enzyme conjugate converts the oxidized nicotinamide adenine dinucleotide (NAD) in the Antibody Reagent to NADH and the change in the absorbance can be measured spectrophotometrically at 340 nm. Enzyme activity decreases upon binding to the antibody, allowing analyte concentrations in a sample to be measured in terms of G6PDH activity.

[0074] Testing is carried out using the SYVAD30-R analyzer Syva-30R, S/N A3562011, available from Siemens Healthcare Diagnostics Inc., Newark Del. The instrument is employed using EMIT immunoassay technology. In the embodiment of the EMIT method used herein and discussed in more detail below, competition between ecstasy and/or amphatemine/methamphetamine analogs on G6PDH conjugates and free drugs in patient samples for antibody binding sites is utilized to determine the amount of ecstasy and/or amphetamine/methamphetamine in patient samples. The enzymatic activity of the free conjugate is measured and is directly proportional to the amount of drugs in the patient sample.

[0075] EMITAssay Results:

[0076] Conclusion: Multi-hapten G6PDH conjugates (7, 9, 10) were successfully prepared using mutant G6PDH with both thiol and amine chemistry. The multi-hapten conjugates (7, 9) were used to produce response curves for the multianalyte of Amphetamine, Methamphetamine, MDMA, MDE and MDEA. The response for each drug can be modulated by changing the conjugate ratios. This concept can be applied to any multi-analyte detection format including EMITformat.

[0077] Experimental Section:

[0078] Reagents

[0079] 1) Antibody Reagent: The amphetamine, methamphetamine and Ecstasy antibodies were formulated in the antibody reagent 1 diluent by spiking at concentrations of 45 ug.Math.mL, 7 ug/mL and (1:250) respectively.

[0080] 2) Conjugate reagent 2: Conjugate lot #7 and #9 (50:50) were formulated separately in the reagent 1 diluent at an Rmax of 750 mA/min. The conjugate solutions were mixed at different ratios (Vol:Vol).

[0081] 3) Standards/Calibrators: Amphetamines, Methamphetamines, Ecstasy (MDMA), MDA and MDEA standards were prepared at concentrations of 0-1000 ng/mL

[0082] 4) Protocol:

Antibody reagent 1=210 uL,

Conjugate Reagent 2=90 uL

[0083] Sample size=5 uL

First Wavelength: 340 nm

[0084] Second Wavelength: 412 nm

[0085] Instrument: Syva-30-R analyzer S/N A3562011

[0086] Preparation of mutant G6PDH (1)

[0087] The mutant G6PDH (1) were invented and prepared as described in references 1-3.

[0088] Preparation of mutant MDA-G6PDH Conjugate (3)

[0089] The hapten (2) and its mutant G6PDH Conjugate (3) were prepared as described in reference 4.

[0090] Preparation of MDA/Amphetamine Mutant G6PDH Conjugate (7)

[0091] Functionization of G6PDH Conjugate (3)

[0092] MDA-G6PDH conjugate (3) in pH 7.0 sodium phosphate buffer (5 mL at 1 mg/mL) is buffer exchanged in a stirred ultrafiltration cell (10 mL) with 55 mM Tris.HCl, pH 8.0 buffer three times. Final enzyme collected is 1.0 mL with a concentration of 5.0 mg/mL. To buffer exchanged G6pDH conjugate in 4 C. are added D-glucose 6-phosphate disodium salt hydrate (Sigma, G7250, 30 mg) and NADH (-NADH disodium salt, USB, Cat. 15345, 30 mg), pH of the solution is checked and found to be 8. To this solution is added 50 L succinimidyl bromoacetate (4, Molecular Bioscience, Cat. 22080, mw: 237.0, 5 mg/mL in DMF) in an ice-water bath. The enzyme solution is stirred in cold room for 45 minutes then dialyzed with 1 L of 55 mM Tris HCl, pH 8.0 buffer, exchanged with fresh buffer five times with three hours between each fresh buffer. The dialyzed enzyme solution (5) collected is 2.1 mL with a concentration of 2.45 mg/mL.

[0093] Reduction of Amphetamine Hapten Linker with TCEP

[0094] 30 mL of carbitol (Diethylene glycol monoethyl ether, Sigma, D1265-1L) and 20 mL of 20 mM of Sodium acetate pH 4.5 buffer are degassed by bubbling nitrogen through each solution for 15 minutes before the addition of amphetamine hapten. To amphetamine hapten (MW 314.47, 10.4 mg) in a small flask are added degassed carbitol (0.4368 ml) and sodium actate (20 mM, pH 4.5, 0.2184 mL). The reaction mixture is stirred under nitrogen atmosphere. TCEP HCl (Sigma, C4706, MW 286.65, 11.8 mg, 1.24 equivalent of amphetamine hapten) is added into amphetamine hapten solution. The progress of the reaction is monitored by TLC (10.5 ml CH.sub.2Cl.sub.2/4 mL MeOH/0.25 mL acetic acid) and product (6) is a spot less polar than that of amphetamine hapten and stained bright yellow with Ellman's Reagent. The reaction finishes within one hour.

[0095] MDA G6PDH Conjugatate with Amphetamine

[0096] MDA-G6PDH deactivated conjugate (5) is bubbled with nitrogen for 30 minutes in an ice-water bath, amphetamine-SH hapten (6, 117 L) is added into the conjugate in an ice water bath drop wise through a syringe under nitrogen atmosphere. The reaction mixture is stirred in a cold room overnight. MDA-G6PDH-amphetamine conjugate is loaded onto a pre-equilibrated G-50 Sephadex column with 55 mM Tris HCl buffer (0.1% NaN.sub.3, pH 8.0), eluted with same buffer. Fractions containing the conjugate (7) are pooled and concentration of the protein is determined by UV at 280 nm. The concentration of the conjugate (7) is determined to be 0.43 mg/ml (11.7 mL) by absorbance at 280 nm.

[0097] Preparation of MDA/Methamphetamine Mutant G6PDH Conjugate (9):

[0098] Functionization of G6PDH Conjugate (3)

[0099] MDA-G6PDH conjugate (3) in pH 7.0 sodium phosphate buffer (4.6 mL at 1 mg/mL) is buffer exchanged in a stirred ultra-filtration cell (10 mL) with 55 mM Tris.HCl, pH 8.0 buffer three times. Final enzyme collected is 0.9 mL with a concentration of 5.0 mg/mL. To buffer exchanged G6pDH conjugate in 4 C. are added D-glucose 6-phosphate disodium salt hydrate (Sigma, G7250, 27.6 mg) and NADH (-NADH disodium salt, USB, Cat. 15345, 27.6 mg), pH of the solution is checked and found to be around 8. To this solution is added 46.0 L succinimidyl bromoacetate (4, Molecular Bioscience, Cat. 22080, mw: 237.0, 5 mg/mL in DMF) in an ice-water bath. The enzyme solution is stirred in cold room (4 C.) for 45 minutes and dialyzed with 1 L of 55 mM Tris HCl, pH 8.0 buffer, exchanged with fresh buffer five times with three hours between each fresh buffer. Dialyzed enzyme solution (5) collected is 2.5 mL with a concentration of 2.44 mg/mL.

[0100] Reduction of Methyl Amphetamine Hapten Linker with TCEP

[0101] 30 mL of carbitol (Diethylene glycol monoethyl ether, Sigma, D1265-1L) and 20 mL of 20 mM of Sodium acetate pH 4.5 buffer are degassed by bubbling nitrogen through for 15 minutes before the addition of methyl amphetamine hapten. To methyl amphetamine hapten (MW 360.58, 11.9 mg) in a small round bottomed flask are added degassed carbitol (0.4998 ml) and sodium acetate (20 mM, pH 4.5, 0.2499 mL). The reaction mixture is stirred under nitrogen atmosphere. TCEP HCl (Sigma, C4706, MW 286.65, 12.77 mg, 1.35 equivalent of methyl amphetamine hapten) is added into the methyl amphetamine hapten solution. The progress of the reaction is monitored by TLC (10.5 ml CH.sub.2Cl.sub.2/4 mL MeOH/0.25 mL acetic acid) and product (8) is a spot less polar than that of methyl amphetamine hapten and stained bright yellow with Ellman's Reagent. The reaction finishes in one hour.

[0102] MDA G6PDH Conjugate with Methyl amphetamine

[0103] MDA-G6PDH deactivated conjugate (5, 2.25 mL) is bubbled with nitrogen for 30 minutes in an ice-water bath, methyl amphetamine-SH hapten (8, 155 L) is added into the conjugate in an ice water bath drop wise through a syringe under nitrogen atmosphere. The reaction mixture is stirred in a cold room (4 C.) overnight. MDA-G6PDH-methyl amphetamine conjugate is loaded onto a pre-equilibrated G-50 Sephadex column with 55 mM Tris HCl buffer (0.1% NaN.sub.3, pH 8.0), eluted with same buffer. Fractions containing the conjugate (9) are pooled and the concentration of the protein is determined to be 0.48 mg/ml (8.88 ml) by UV at 280 nm.

[0104] Preparation of MDA/Amphetamine/Methamphetamine Mutant G6PDH Conjugate (10):

[0105] Functionization of G6PDH Conjugate (3)

[0106] MDA-G6PDH conjugate (3) in sodium pH 7.0 phosphate buffer (15 mL at 1 mg/mL) is buffer exchanged in a stirred ultra-filtration cell (50 mL) with 55 mM Tris.HCl, pH 8.0 buffer three times. Final enzyme collected is 3.0 mL with a concentration of 5.0 mg/mL. To buffer exchanged G6pDH conjugate in 4 C. are added D-glucose 6-phosphate disodium salt hydrate (Sigma, G7250, 90.2 mg) and NADH (-NADH disodium salt, USB, Cat. 15345, 90.1 mg), pH of the solution is checked and found to be around 8. To this G6PDH conjugate solution is added 150.0 L succinimidyl bromoacetate (4, Molecular Bioscience, Cat. 22080, mw: 237.0, 5 mg/mL in DMF) in an ice-water bath. The solution is stirred in cold room (4 C.) for 45 minutes. The enzyme solution is dialyzed with 1 L of 55 mM Tris HCl, pH 8.0 buffer, exchanged with fresh buffer five times with three hours between each fresh buffer. Dialyzed enzyme solution (5) collected is 6.7 mL with a concentration of 4.52 mg/mL.

[0107] Reduction of Methyl Amphetamine/Amphetamine Hapten Linkers with TCEP

[0108] 30 mL of carbitol (Diethylene glycol monoethyl ether, Sigma, D1265-1L) and 30 mL of 20 mM of Sodium acetate pH 4.5 buffer are degassed by bubbling nitrogen through for 15 minutes before the addition of hapten. To methyl amphetamine hapten (MW 360.58, 10.4 mg) in a small round bottomed flask are added degassed carbitol (0.4368 ml) and sodium acetate (20 mM, pH 4.5, 0.2184 mL). The reaction mixture is stirred under nitrogen atmosphere. TCEP HCl (Sigma, C4706, MW 286.65, 11.16 mg, 1.35 equivalent of methyl amphetamine hapten) is added into methyl amphetamine hapten solution. The progress of the reaction is monitored by TLC (10.5 ml CH.sub.2Cl.sub.2/4 mL MeOH/0.25 mL acetic acid) and product (8) is a spot less polar than that of methyl amphetamine hapten and stained bright yellow with Ellman's Reagent. The reaction finishes in one hour. At the same time, to amphetamine hapten (MW 314.47, 7.5 mg) in a small round bottomed flask are added degassed carbitol (0.3150 mL) and NaOAc (20 mM, pH 4.5, 0.1575 mL). The solution is stirred under nitrogen atmosphere; TCEP HCl (Sigma, C4706, MW 286.65, 9.23 mg, 1.35 equivalent of amphetamine hapten) is added into amphetamine hapten solution. The progress of the reaction is monitored by TLC (10.5 ml CH.sub.2Cl.sub.2/4 mL MeOH/0.25 mL acetic acid) and product (6) is a spot less polar than that of methyl amphetamine hapten and stained bright yellow with Ellman's Reagent. The reaction finishes in one hour.

[0109] MDA G6PDH Conjugate with Methyl amphetamine and Amphetamine

[0110] MDA-G6PDH deactivated conjugate (5, 1.8 mL, 4.52 mL) is bubbled with nitrogen for 30 minutes in an ice-water bath, methyl amphetamine-SH hapten (8, 65.5 L) and amphetamine-SH hapten (6, 57.1 L) (molar ratio of methyl amphetamine hapten and amphetamine hapte is 1 to 1) are added into the MDA G6PDH deactivated conjugate in an ice water bath drop wise through a syringe under nitrogen atmosphere. The reaction mixture is stirred in a cold room overnight. MDA-G6PDH-methyl amphetamine/methyl amphetamine conjugate is loaded onto a pre-equilibrated G-50 Sephadex column with 55 mM Tris HCl buffer (0.1% NaN.sub.3, pH 8.0), eluted with same buffer. Fractions containing the conjugate (10) are pooled and concentration of the protein is determined to be 0.44 mg/ml (11.8 ml) by UV at 280 nm.

REFERENCES

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