EP0312248A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Acridinium ester labelling and purification of nucleotide probes
Assignee: Gen Probe Inc
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Filing: 1988-10-05
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Granted: —
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Expires: 2008-10-05
Abstract
Abstract Methods for the construction, labelling and subsequent purification of nucleic acid probes containing primary amines with acridinium esters 4-(2-succinimidyloxycarbonyl-ethyl)phenyl10-methylacridinium-9-carboxylate fluorosulfonate). The method for attaching acridinium esters to probes uses high (0.1 to 50mM) acridinium ester concentrations achieved using organic solvent in concentrations of 20% to 80% by volume, and may be carried out either in solution, or with one or the other of the acridinium ester or the probe suspended in solution. Purification (the separation of labelled probe from unlabelled probe and free label) involves (1) first removing most of the free acridinium ester label from probe using rapid separation techniques, then (2) removing substantially all remaining free label from the probe and separating labelled probe from unlabelled probe, involves specific applications of ion exchange, reverse phase or hydroxyapatite HPLC.
Inventors
- 👤 Lyle John Arnold
- 👤 Norman Charles Nelson
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-06 12:05
CPC Classifications
C; C07; C07H; C07H21/00; C07H21/02
Claims Analysis
Total Claims: 28
Claim 1
Independent Claim
59 words
A method of labelling a nucleic acid probe having a first conjugation moiety, with an acridinium ester labelling reagent having a second conjugation moiety, including the step of: combining said labelling reagent and said nucleic acid probe so as to effectuate a labelling reagent concentration of approximately 0.1-50mM to obtain acridinium ester labelled nucleic acid probes in high yield.
Claim 2
Independent Claim
59 words
A method of labelling a nucleic acid probe having a first conjugation moiety, with an acridinium ester labelling reagent having a second conjugation moiety, including the step of: combining said labelling reagent and said nucleic acid probe so as to effectuate a labelling reagent concentration of approximately 0.1-50mM to obtain acridinium ester labelled nucleic acid probes in high yield.
Claim 3
Independent Claim
74 words
A method for labelling a nucleic acid probe having a first conjugation moiety, with an acridinium ester labelling reagent having a second conjugation moiety, including the step of: combining said labelling reagent and said nucleic acid probe in a manner so as to effectuate a labelling reagent concentration of approximately 0.1-50mM and an organic solvent concentration of approximately 20% to 80% by volume to obtain acridinium ester labelled nucleic acid probes in high yield.
Claim 4
Independent Claim
74 words
A method for labelling a nucleic acid probe having a first conjugation moiety, with an acridinium ester labelling reagent having a second conjugation moiety, including the step of: combining said labelling reagent and said nucleic acid probe in a manner so as to effectuate a labelling reagent concentration of approximately 0.1-50mM and an organic solvent concentration of approximately 20% to 80% by volume to obtain acridinium ester labelled nucleic acid probes in high yield.
Claim 5
Dependent Claim
39 words
The method of claim 2 wherein said organic solvent is DMSO, CH₃CN, dimethylformamide, dioxane, acetone or methanol. 4. A method as in any one of the preceding claims wherein the step is carried out in the pH range 7-9.
Claim 6
Independent Claim
21 words
A method as in any one of the preceding claims wherein the step is carried out in the temperature range 15-40°C.
Claim 7
Independent Claim
18 words
A method as in any one of the preceding claims wherein the step is carried out in solution.
Claim 8
Independent Claim
32 words
A method as in any one of the preceding claims wherein the step is carried out with said nucleic acid probe in solution and wherein said labelling reagent is suspended in solution.
Claim 9
Independent Claim
32 words
A method as in any one of claims 1 to 6 wherein the step is carried out with said labelling reagent in solution and said nucleic acid probe is suspended in solution.
Claim 10
Independent Claim
65 words
A method for labelling nucleic acid probes having a first conjugation moiety with an acridinium ester labelling reagent having a second conjugation moiety, including the steps of: combining said labelling reagent in approximately a 0.1-50mM concentration; with an organic solvent in concentration of approximately 20% to 80% by volume to obtain acridinium ester labelled nucleic acid probes in high yield; quenching the unreacted labelling reagent.
Claim 11
Independent Claim
65 words
A method for labelling nucleic acid probes having a first conjugation moiety with an acridinium ester labelling reagent having a second conjugation moiety, including the steps of: combining said labelling reagent in approximately a 0.1-50mM concentration; with an organic solvent in concentration of approximately 20% to 80% by volume to obtain acridinium ester labelled nucleic acid probes in high yield; quenching the unreacted labelling reagent.
Claim 12
Independent Claim
154 words
A method for labelling nucleic acid probes having a first conjugation moiety with an acridinium ester labelling reagent having a second conjugation moiety, including the steps of: combining said labelling reagent in approximately a 0.1-50mM concentration with an organic solvent in a 20% to 80% concentration by volume to obtain acridinium ester labelled nucleic acid probes in high yield, wherein said first and second conjugation moieties are selected, respectively from the group of pairs consisting of: (1) in pH range 7-9; (2) in pH range 8-10; (3) in pH range 7-9; (4) - NH₂ , SCN- in pH range 7-9; (5) in pH range 7-9; (6) in pH range 7-9; (7) in pH range 6-8; (8) in pH range 6-8; (9) in pH range 6-9; (10) in pH range 5-9. with an organic solvent in concentration of approximately 20% to 80% by volume to obtain acridinium ester labelled nucleic acid probes in high yield.
Claim 13
Independent Claim
154 words
A method for labelling nucleic acid probes having a first conjugation moiety with an acridinium ester labelling reagent having a second conjugation moiety, including the steps of: combining said labelling reagent in approximately a 0.1-50mM concentration with an organic solvent in a 20% to 80% concentration by volume to obtain acridinium ester labelled nucleic acid probes in high yield, wherein said first and second conjugation moieties are selected, respectively from the group of pairs consisting of: (1) in pH range 7-9; (2) in pH range 8-10; (3) in pH range 7-9; (4) - NH₂ , SCN- in pH range 7-9; (5) in pH range 7-9; (6) in pH range 7-9; (7) in pH range 6-8; (8) in pH range 6-8; (9) in pH range 6-9; (10) in pH range 5-9. with an organic solvent in concentration of approximately 20% to 80% by volume to obtain acridinium ester labelled nucleic acid probes in high yield.
Claim 14
Independent Claim
55 words
A method for labelling nucleic acid probes having a first conjugation moiety with an N-hydroxysuccinimide-acridinium ester labelling reagent, comprising the step of combining said labelling reagent in approximately a 1-10mM concentration with an organic solvent in concentration of approximately 20% to 80% by volume, to obtain acridinium ester labelled nucleic acid probes in high yield.
Claim 15
Independent Claim
55 words
A method for labelling nucleic acid probes having a first conjugation moiety with an N-hydroxysuccinimide-acridinium ester labelling reagent, comprising the step of combining said labelling reagent in approximately a 1-10mM concentration with an organic solvent in concentration of approximately 20% to 80% by volume, to obtain acridinium ester labelled nucleic acid probes in high yield.
Claim 16
Independent Claim
56 words
A method for labelling nucleic acid probes having a first conjugation moiety with an acridinium ester-methoxyimidate labelling reagent, comprising the step of combining said labelling reaGent in approximately a 1-10 mM concentration wlth an organic solvent in concentration of approximately 20% to 80% by volume, to obtain acridinium ester labelled nucleic acid probes in high yield.
Claim 17
Independent Claim
56 words
A method for labelling nucleic acid probes having a first conjugation moiety with an acridinium ester-methoxyimidate labelling reagent, comprising the step of combining said labelling reaGent in approximately a 1-10 mM concentration wlth an organic solvent in concentration of approximately 20% to 80% by volume, to obtain acridinium ester labelled nucleic acid probes in high yield.
Claim 18
Independent Claim
83 words
A method for separating acridinium ester-labelled probe from unlabelled probe and from free acridinium ester label to a high degree of purity, said method comprising: binding said labelled probe to an HPLC column from the group consisting of ion exchange, reverse phase and hydroxyapatite, in a first buffer; contacting said HPLC column with a second buffer or a gradient of said first buffer and said second buffer, so as to effectuate differential elution of labelled probe, unlabelled probe and free acridinium ester label.
Claim 19
Independent Claim
83 words
A method for separating acridinium ester-labelled probe from unlabelled probe and from free acridinium ester label to a high degree of purity, said method comprising: binding said labelled probe to an HPLC column from the group consisting of ion exchange, reverse phase and hydroxyapatite, in a first buffer; contacting said HPLC column with a second buffer or a gradient of said first buffer and said second buffer, so as to effectuate differential elution of labelled probe, unlabelled probe and free acridinium ester label.
Claim 20
Dependent Claim
37 words
The method of claim 13 wherein said column is ion exchange and wherein said first buffer is 20mM NaOAc, pH 5.5, 20% CH₃CN, and wherein said second buffer is 20mM NaOAc, pH 5.5, 20% CH₃CN, 1M LiCl.
Claim 21
Dependent Claim
46 words
The method of claim 13 wherein said column is ion exchange and wherein said gradient is from a first buffer consisting of 20mM NaOAc, pH 5.5, 20% CH₃CN, 0.45 M LiCl to a second buffer consisting of 20mM NaOAc, pH 5.5, 20% CH₃CN, 0.7 M LiCl.
Claim 22
Dependent Claim
30 words
The method of claim 13 wherein said column is reverse phase and wherein said first buffer is 0.1 M triethylammonium acetate, pH 7.0 and wherein said second buffer is CH₃CN.
Claim 23
Dependent Claim
43 words
The method of claim 13 wherein said column is reverse phase and wherein said gradient is from a first buffer consisting of 0.1 M triethylammonium acetate, pH 7.0, 10% CH₃CN to a second buffer consisting of 0.1M triethylammonium acetate, pH 7.0, 50% CH₃CN.
Claim 24
Independent Claim
73 words
A method for separating acridinium ester labelled prob from unlabelled probe and from free acridinium ester label to a high degree of purity, said method comprising: removing most of said free acridinium ester label from said labelled probe using a rapid separation means; removing substantially all remaining free acridinium ester label from probe and separating labelled probe from unlabelled probe using HPLC selected from the group consisting of ion-exchange, reverse phase or hydroxyapatite.
Claim 25
Independent Claim
73 words
A method for separating acridinium ester labelled prob from unlabelled probe and from free acridinium ester label to a high degree of purity, said method comprising: removing most of said free acridinium ester label from said labelled probe using a rapid separation means; removing substantially all remaining free acridinium ester label from probe and separating labelled probe from unlabelled probe using HPLC selected from the group consisting of ion-exchange, reverse phase or hydroxyapatite.
Claim 26
Dependent Claim
88 words
19 . The method of claim 18 wherein said rapid selection means is selected from the group consisting of: (1) nucleic acid precipitation; (2) binding of free label to hydroxyapatite, Bio-Beads SM2, Sep-pak or solid support to which label binds and either free labelled probe does not, or to which labelled probe binds and free label does not; (3) rapid gel filtration; (4) extraction of free acridinium ester label into an organic phase; (5) filtration; (6) rapid ion-exchange chromatography; (7) ion exchange HPLC; and (8) reverse phase HPLC.
Claim 27
Dependent Claim
63 words
20 . The method of claim 19 wherein said rapid separation means is ethanol precipitation and wherein said HPLC is ion exchange and wherein said removal and separation is achieved using a gradient from a first buffer consisting of 20mM NaOAc, pH 5.5, 20% CH₃CN, 0.45 M LiCl to a second buffer consisting of 20mM NaOAc, pH 5.5, 20% CH₃CN, 0.7 M LiCl.
Claim 28
Dependent Claim
59 words
The method of claim 19 wherein said rapid separation means is ethanol precipitation and wherein said HPLC is reverse phase and wherein said removal and separation is achieved using a gradient from a first buffer consisting of 0.1 M triethylammonium acetate, pH 7.0, 10% CH₃CN to a second buffer consisting of 0.1 M triethylammonium acetate, pH 7.0, 50% CH₃CN.
References Cited (Backward Citations)
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