US5223402A — Method of detecting microbes utilizing chemilumin…

Claim 1

Independent Claim 124 words

A method of detecting hydrolase activity of biologically active microorganisms in a sample, said method including the steps of: combining the sample with 1,2-dioxetane compound (1,2-dc), which decomposes to a light-emitting species by at least one hydrolytic enzyme of the microorganisms in the sample; triggering light emission from the 1,2-dc by said at least one hydrolytic enzyme initiating decomposition of the 1,2-dc; and detecting and integrating the light emission over an extended period of time as an indication of the presence of the hydrolase activity of the microorganisms in the sample by exposing a light-sensitive detector to the emitted light over a period of time, the detector summing the absolute value of the emitted light over time to indicate the presence of the microorganisms.

Claim 2

Independent Claim 124 words

A method of detecting hydrolase activity of biologically active microorganisms in a sample, said method including the steps of: combining the sample with 1,2-dioxetane compound (1,2-dc), which decomposes to a light-emitting species by at least one hydrolytic enzyme of the microorganisms in the sample; triggering light emission from the 1,2-dc by said at least one hydrolytic enzyme initiating decomposition of the 1,2-dc; and detecting and integrating the light emission over an extended period of time as an indication of the presence of the hydrolase activity of the microorganisms in the sample by exposing a light-sensitive detector to the emitted light over a period of time, the detector summing the absolute value of the emitted light over time to indicate the presence of the microorganisms.

Claim 3

Dependent Claim 25 words

A method as set forth in claim 1 further including the step of enriching the amount of the at least one enzyme in the sample.

Claim 4

Dependent Claim 27 words

A method as set forth in claim 2 wherein the enriching step is further defined as enhancing enzyme synthesis by the microorganisms prior to said combining step.

Claim 5

Dependent Claim 26 words

A method as set forth in claim 2 wherein said enriching step is further defined as enhancing enzyme synthesis by the microorganisms after said combining step.

Claim 6

Independent Claim 75 words

A method as set forth in claims 3 or 4 wherein said enhancing step is further defined as incubating the sample for one to six hours in a minimal medium containing cations normally required by alkaline phosphatase as cofactors, a carbon source, a nitrogen source, and a scavenger of free inorganic phosphates, the medium lacking inorganic phosphate and being buffered to an alkaline pH, which optimizes enzyme synthesis and prevents repressing by the end product.

Claim 7

Dependent Claim 50 words

A method as set forth in claim 2 wherein said detecting and integrating steps further include the step of changing the pH of the media to optimize light emission from the 1-2-dioxetane compound that was initially decomposed by said at least one hydrolytic enzyme in the sample to produce chemiluminescence.

Claim 8

Dependent Claim 43 words

A method as set forth in claim 6 wherein said incubating and changing steps are further defined as incubating the sample in media initially at a neutral pH and changing the pH of the media to an alkaline pH to optimize light emission.

Claim 9

Dependent Claim 24 words

A method as set forth in claim 1 further including the step of releasing intracellular enzyme of the microorganisms prior to said combining step.

Claim 10

Dependent Claim 43 words

A method as set forth in claim 8 wherein the microorganisms are gram negative bacteria, said releasing step being further defined as removing an outer membrane from the bacteria and releasing the at least one enzyme from the periplasmic space of the bacteria.

Claim 11

Dependent Claim 28 words

A method as set forth in claim 8 wherein the microorganisms are gram positive bacteria, said releasing step being further defined as exposing the bacteria to a detergent.

Claim 12

Dependent Claim 26 words

A method as set forth in claim 8 wherein the microorganisms are gram positive bacteria, said releasing step being further defined as osmotically shocking the bacteria.

Claim 13

Dependent Claim 26 words

A method as set forth in claim 8 wherein the microorganisms are gram positive bacteria, said releasing step being further defined as sonically lysing the bacteria.

Claim 14

Dependent Claim 34 words

A method as set forth in claim 8 wherein the microorganisms are gram positive bacteria, said releasing step being further defined as exposing the bacteria to lysozyme or other bacterial cell wall lytic enzymes.

Claim 15

Dependent Claim 32 words

A method as set forth in claim 1 wherein said detecting and integrating steps are further defined as detecting and integrating light emission over a period of time greater than two minutes.

Claim 16

Dependent Claim 30 words

A method as set forth in claim 14 wherein said detecting step is further defined as detecting and integrating light emission over a period between one minute and two hours.

Claim 17

Independent Claim 106 words

A method of identifying hydrolase activity from biologically active microorganisms in biological and non-biological samples, said method including the steps of: combining the sample with a 1-2-dioxetane compound (1,2-dc); hydrolytic enzymes of the microorganisms in the sample initiating decomposition of the 1,2-dc which decomposes to a light-emitting species; and detecting and integrating the light emission over an extended period of time as an indication of the identity of the microorganisms in the sample by exposing a light-sensitive detector to the emitted light over a period of time, the detector summing the absolute value of the emitted light over time to indicate the presence of the microorganisms.

Claim 18

Independent Claim 106 words

A method of identifying hydrolase activity from biologically active microorganisms in biological and non-biological samples, said method including the steps of: combining the sample with a 1-2-dioxetane compound (1,2-dc); hydrolytic enzymes of the microorganisms in the sample initiating decomposition of the 1,2-dc which decomposes to a light-emitting species; and detecting and integrating the light emission over an extended period of time as an indication of the identity of the microorganisms in the sample by exposing a light-sensitive detector to the emitted light over a period of time, the detector summing the absolute value of the emitted light over time to indicate the presence of the microorganisms.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1996-06-28	1996-12-28	1997-06-29	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2000-06-28	2000-12-28	2001-06-29	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2004-06-28	2004-12-28	2005-06-29	$7700.00	Window Closed / Lapsed

Method of detecting microbes utilizing chemiluminescent compound

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