Patent EP0059645A1 successfully refreshed from Google Patents.
EP0059645A1
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EP
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Method and kit for identification of beta-lactamases
Assignee: National Research Development Corp UK
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Filing: 1982-03-03
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Granted: —
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Expires: 2002-03-03
Abstract
Abstract The invention provides a way of identifying the beta-lactamase specifically, by monitoring competition reactions between a chromogenic substrate, for example nitrocefin, and various competing substrates, preferably some of the newer beta-lactams, for example cefoxitin, moxalactam, MK 0787 etc. Detailed study of these reactions by the inventor has shown unexpectedly marked variations in the reactivity (affinity) of these enzymes for the different beta-tactams. On this basis the invention provides an assay for the identifying "unknown" beta-lactamases based on comparing their behaviour in the competition reaction with affinity data on known beta-lactamases. The chromogenic substrate and a selection of the most useful beta-lactams are provided as a kit. It can contain prepared mixtures of the beta-lactams and chromogenic substrate. The assay can be carried out visually using micro-titre plates and a simple scoring system or more quantitatively by spectrophotometric scanning. It can be used for the rapid identification of beta-lactamases in a hospital laboratory or for the identification of gram-negative bacteria based on the beta-lactamases which they produce.
Inventors
- 👤 Richard James
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-09 00:53
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/02; C12Q1/04; C12Q1/18; C12Q1/34; G; G01; G01N; G01N33/00; G01N33/02; G01N33/04; G01N2333/00; G01N2333/90; G01N2333/914; G01N2333/978; G01N2333/986; G01N2415/00
Claims Analysis
Total Claims: 40
Claim 1
Independent Claim
130 words
A method of identifying or helping to identify an unidentified beta-lactamase, which method comprises performing a competitive assay on said beta-lactamase with (1) a chromogenic beta-lactam-containing substrate capable of undergoing a change in its visible light spectrum when reacted under the competitive assay conditions with beta-lactamases and (2) a competing substrate, monitoring the assay for a change in the visible light spectrum of the chromogenic substrate, thence determining a relative affinity of the competing substrate to the chromogenic substrate for the unidentified beta-lactamase, if necessary repeating said assay at least once using for each assay a different competing substrate, or different conditions of assay, and identifying the beta-lactamase by reference to a compilation of previously determined relative affinities of each competing substrate relative to the chromogenic substrate for different beta-lactamases.
Claim 2
Independent Claim
130 words
A method of identifying or helping to identify an unidentified beta-lactamase, which method comprises performing a competitive assay on said beta-lactamase with (1) a chromogenic beta-lactam-containing substrate capable of undergoing a change in its visible light spectrum when reacted under the competitive assay conditions with beta-lactamases and (2) a competing substrate, monitoring the assay for a change in the visible light spectrum of the chromogenic substrate, thence determining a relative affinity of the competing substrate to the chromogenic substrate for the unidentified beta-lactamase, if necessary repeating said assay at least once using for each assay a different competing substrate, or different conditions of assay, and identifying the beta-lactamase by reference to a compilation of previously determined relative affinities of each competing substrate relative to the chromogenic substrate for different beta-lactamases.
Claim 3
Dependent Claim
136 words
A method according to Claim 1, using a kit comprising:- (a) a chromogenic substrate (b) competing substrates, each of which will distinguish between the beta-lactamases or groups of beta-lactamases specified in at least one of the following pairings (1) to (7):- (1) the group consisting of TEM-1, TEM-2 and SHV-1 from the group consisting of HMS-1, OXA-1, OXA-2, OXA-3, PSE-1, PSE-2, PSE-3 and PSE-4; (2) SHV-1 from the group consisting of TEM-1 and TEM-2; (3) the group consisting of PSE-1, PSE-2, PSE-3 and PSE-4 from the group consisting of HMS-1, OXA-1, OXA-2 and OXA-3; (4) the group consisting of PSE-2 and PSE-3 from the group consisting of PSE-1 and PSE-4; (5) PSE-2 from PSE-3; (6) HMS-1 from the group consisting of OXA-1, OXA-2 and OXA-3; and (7) OXA-1 from the group consisting of OXA-2 and OXA-3.
Claim 4
Dependent Claim
66 words
A method according to Claim 2, modified in that pairing (3) is replaced by the following two pairings, namely (3A) the group consisting of PSE-1, PSE-3 and PSE-4 from the group consisting of PSE-2, HMS-1, OXA-1, OXA-2 and OXA-3 (3B) PSE-2 from the group consisting of HMS-1, OXA-1, OXA-2 and OXA-3, in that PSE-2 is omitted from pairing (4) and in that pairing (5) is omitted.
Claim 5
Dependent Claim
83 words
A method according to Claim 2 or 3, modified in that pairing (1) is replaced by two pairings, namely (lA) the group consisting of TEM-l, TEM-2, SHV-1 and OXA-1, from the group consisting of HMS-1, OXA-2, OXA-3, PSE-1, PSE-2, PSE-3 and PSE-4; and (1B) OXA-1 from the group consisting of TEM-1, TEM-2 and SHV-1, in that OXA-1 is omitted from pairings (3) and (6) or, in the case of Claim 3, pairings (3A), (3B) and (6) and in that pairing (7) is omitted.
Claim 6
Dependent Claim
47 words
A method according to Claim 2, 3 or 4, wherein the kit comprises a competing substrate which distinguishes the group consisting of E. cloacae and ampicillin-induced Pseudomonas aeruginosa 1937E beta-lactamases from the group consisting of TEM-1, TEM-2, SHV-1, HMS-1, OXA-1, OXA-2, OXA-3, PSE-1, PSE-2, PSE-3 and PSE-4.
Claim 7
Dependent Claim
26 words
A method according to Claim 5, wherein the kit comprises a competing substrate which distinguishes the E, cloacae beta-lactamase from the ampicillin-induced Pseudomonas aeruginosa 1937E beta-lactamase.
Claim 8
Dependent Claim
24 words
A method according to Claim 2, 3, 4, 5 or 6, wherein the kit comprises a competing substrate which distinguishes between OXA-2 and OXA-3.
Claim 9
Dependent Claim
25 words
A method according to Claim 2, 3, 4, 5, 6 or 7, wherein the kit comprises a competing substrate which distinguishes between PSE-1 and PSE-4.
Claim 10
Independent Claim
13 words
A method according to any preceding claim wherein the chromogenic substrate is nitrocefin.
Claim 11
Independent Claim
37 words
A method according to any preceding claim wherein the relative affinity is determined quantitatively and each assay is carried out in a manner which enables irreversible inhibition of the beta-lactamase by the competing substrate to be detected.
Claim 12
Dependent Claim
53 words
A method according to Claim 10 wherein each assay is carried out using at least 4 different ratios of competing substrate to chromogenic substrate and a relative affinity is determined as the ratio:- Concentration of beta-lactam-containing substrate which gives a defined percentage inhibition of cleavage of the chromogenic substrate Concentration of chromogenic substrate
Claim 13
Dependent Claim
13 words
A method according to Claim 11 wherein the defined percentage is 50 percent.
Claim 14
Dependent Claim
15 words
A method according to Claim 10, 11 or 12 wherein the assay is monitored spectrophotometrically.
Claim 15
Dependent Claim
85 words
A method according to Claim 13 carried out using a kit defined in any one of Claims 2 to 8, in which the competing substrates of the kit will distinguish between the beta-lactamases or groups thereof by a ratio of at least 2.5:1 in their relative substrate affinity index defined by the ratio:- Concentration of beta-lactam-containing substrate which gives 50% inhibition of cleavage of the chromogenic substrate. Concentration of the chromogenic substrate or will distinguish them according to whether or not they give irreversible inhibition.
Claim 16
Dependent Claim
14 words
A method according to Claim 14 wherein the distinguishing ratio is at least 10:1.
Claim 17
Dependent Claim
93 words
A method according to Claim 13, carried out using a kit which comprises:- (a) nitrocefin and (b) competing substrates selected from each of the following classes (1) to (7) (1) Cefoxitin, cefotaxime, moxalactam, ceftizoxime and RO-13-9904; (2) Cefoxitin, moxalactam, RO-13-9904 and MK 0787; (3) SQ 14,359, moxalactam and MK 0787; (4) Cefamandole, cephradine, CP 45,899, clavulanic acid and MK 0787; (5) Cloxacillin, cephaloridine, cefotaxime, SQ 14,359, cefamandole, mecillinam, RO-13-9904 and CP 45,899; (6) Cefoperazone, carbenicillin, mecillinam, ceftazidime and MK 0787; and (7) Cephaloridine, cefoxitin, SQ 14,359, CP 45,899, clavulanic acid,.MK 0787 and ticarcillin.
Claim 18
Dependent Claim
39 words
A method according to Claim 16, modified in that class (3) is replaced by the following classes (3A) and (3B):- (3A) Cefotaxime (3B) Cefamandole, cephradine, MK 0787, CP 45,899 and clavulanic acid and in that class 5 is omitted.
Claim 19
Dependent Claim
43 words
A method according to Claim 16 or 17, modified in that class (1) is replaced by the following classes (lA) and (1B) (lA) SQ 14,359 (1B) moxalactam, cephradine, mecillinam, ceftizoxime, RO-13-9904, CP 45,899 and MK 0787 and in that class (7) is omitted.
Claim 20
Dependent Claim
55 words
A method according to Claim 16, 17 or 18, in which the competing substrates are further selected from any one or more of the following further classes:- (8) cloxacillin, cefotaxime, SQ 14,359, moxalactam, carbenicillin, ceftazidime, clavulanic acid, amoxycillin and ticarcillin; (9) ampicillin, cloxacillin, cefoxitin and cefuroxime; and (10) ampicillin, RO-13-9904, CP 45,899 and clavulanic acid.
Claim 21
Dependent Claim
29 words
A method according to Claim 19, wherein the competing substrates include at least one from class (8) and at least one of: moxalactam, carbenicillin, cefamandole, mecillinam, ceftizoxime and RO-13-9904.
Claim 22
Dependent Claim
191 words
A method according to Claim 13, carried out using a kit which comprises:- (a) nitrocefin as chromogenic substrate and (b) any of the following sets of competing substrates:- (i) cefoxitin, (ii) moxalactam or MK 0787 and (iii) mecillinam; (i) cefoxitin, (ii) MK 0787 and (iii) any one of cloxacillin, cephaloridine, cefotaxime, SQ 14,359, cefamandole, RO-13-9904 and CP 45,899; (i) cefoxitin, (ii) SQ 14,359, (iii) any one of cefamandole, cephradine, CP 45,899, clavulanic acid and MK 0787, and (iv) any one of cefoperazone, carbenicillin, mecillinam or ceftazidime; (i) cefoxitin (ii) cefotaxime and (iii) MK 0787.or (iii) any one of cefamandole, cephradine, CP 45,899, and clavulanic acid and (iv) any one of cefoperazone, carbenicillin, mecillinam and ceftazidime; (i) moxalactam, (ii) MK 0787 and (iii) any one of cloxacillin, cephaloridine, cefotaxime, SQ 14,359, cefamandole, mecillinam, RO-13-9904 and CP 45,899; (i) moxalactam, (ii) CP 45,899 and (iii) any one of cefoperazone, carbenicillin, mecillinam and ceftazidime; (i) moxalactam, (ii) mecillinam and (iii) clavulanic acid; (i) RO-13-9904 and (ii) MK 0787; (i) RO-13-9904, (ii) SQ 14,359, (iii) any one of cefamandole, cephradine, CP 45,899 and clavulanic acid and (iv) any one of cefoperazone, carbenicillin, mecillinam and ceftazidime.
Claim 23
Independent Claim
26 words
A method according to any one of Claims 1 to 9 wherein a relative affinity is determined semi-quantitatively and the assay is monitored by visual inspection.
Claim 24
Dependent Claim
46 words
A method according to Claim 22 wherein each assay is carried out using a ratio of competing substrate to chromogenic substrate such that a visually recognisable difference in the spectrum of the chromogenic substrate occurs between competitive assays of different beta-lactamases using the same competing beta-lactamase.
Claim 25
Dependent Claim
37 words
A method according to Claim 23 wherein the relative affinity is determined as a score according to whether there is substantially no change in the spectrum of the chromogenic substrate, a small change or a large one.
Claim 26
Dependent Claim
17 words
A method according to Claim 22, 23 or 24 wherein the assay is monitored merely by eye.
Claim 27
Dependent Claim
24 words
A method according to Claim 25 carried out using a kit comprising:- (a) nitrocefin as chromogenic substrate and (b) cefoxitin, cefotaxime, cefamandole and mecillinam.
Claim 28
Dependent Claim
41 words
A method according to Claim 25 or 26 wherein the kit includes a beta-lactam selected from at least one of the following groupings (1) to (3):- (1) moxalactam, cloxacillin, cefotaxime or SQ 14,359 (2) cloxacillin, cefoxitin or cefuroxime and (3) RO-13-9904.
Claim 29
Dependent Claim
20 words
A method according to Claim 22, 23 or 24 wherein the assay is monitored spectrophotometrically and irreversible inhibition is determined.
Claim 30
Dependent Claim
43 words
A method according to Claim 28 carried out using a kit comprising:- (a) nitrocefin as chromogenic substrate and (b) either cefoxitin, cefotaxime, cefamadole, mecillinam, moxalactam, cloxacillin, clavulanic acid and RO-13-9904 or SQ 14,359, moxalactam, cephradine, ceftazidime, cloxacillin, cefoxitin, CP 45,899 and clavulanic acid.
Claim 31
Independent Claim
24 words
A method according to any preceding claim wherein the beta-lactamase to be identified is extracted from bacteria obtained from a sample of human blood.
Claim 32
Dependent Claim
57 words
A kit for use in a method according to Claim 10, comprising:- (a) at least two sets of mixtures of the chromogenic substrate and a competing substrate, each in at least 4 different ratios per set and the competing substrate being different in each set of mixtures, and (b) the chromogenic substrate per se, as a control.
Claim 33
Dependent Claim
42 words
A kit according to Claim 31 which further includes a spectrophotometer, a member carrying the compilation of relative affinities and a computer programmed to compute the relative affinities determined from the assay and to compare the determined relative affinities with the compilation.
Claim 34
Dependent Claim
24 words
A kit for use in a method according to Claim 1, said kit being as defined in any one of Claims 1 to 9.
Claim 35
Dependent Claim
24 words
A kit for use in a method according to Claim 16, said kit being as defined in any one of Claims 16 to 22.
Claim 36
Dependent Claim
47 words
A kit for use in a method according to Claim 23, comprising:- (a) at least two sets of a mixture of the chromogenic substrate with a competing substrate, each in a ratio as defined in Claim 23, and (b) the chromogenic substrate per se, as a control.
Claim 37
Dependent Claim
36 words
A kit according to Claim 35 which further includes a member carrying a compilation of relative affinities in the form of a chart of scores as defined in Claim 24 or a colour chart or both.
Claim 38
Dependent Claim
25 words
A kit for use in a method according to Claim 23, said kit being as defined in any one of Claims 26, 27 and 29.
Claim 39
Dependent Claim
17 words
A kit according to Claim 37 containing cefoxitin in two different ratios to nitrocefin, as chromogenic substrate.
Claim 40
Dependent Claim
21 words
A kit according to any one of Claims 31 to 38, which further includes an identified beta-lactamase for testing the kit.
References Cited (Backward Citations)
GB1408391A
Cited
GB2026689A
Cited
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