# US 7,745,136 B1 · Direct quantification of ribosome inactivating protein

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- Number: US 7,745,136 B1
- Document ID: US7745136B1
- Status: Granted
- Kind: B1
- Application number: 12/362,599
- Filed: 2009-01-30
- Granted: 2010-06-29
- Earliest claimed: 2009-01-30
- Assignees: The United States of America as represented by the Secretary of the Army
- Inventors: Russell M. Dorsey
- Web page: https://patentgrep.com/patent/US7745136B1

## CPC

- C12Q 1/6804: Nucleic acid analysis using immunogens (immunoassay G01N33/53)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

A method for detecting ribosome inactivating protein (RIP) activity in a sample is provided that involves contacting a sample that contains an RIP with an inventive substrate that is depurinated to form product. The product is hybridized to a template and cleaved by an apurinic/apyrimidinic endonuclease such that releasing the blocked 3′ end only in a sample that contains RIP activity. The 5′ end of the product serves as a primer for extension by a polymerase reaction. The newly synthesized strand complementary to the template is detected by RT-PCR processes. A kit is provided suitable for field or laboratory use.

## Claims

### Claim 1

A process for detecting activity of a ribosome inactivating protein, comprising:
providing an oligonucleotide substrate consisting of SEQ ID NO: 2 with a GAGA loop;
contacting said substrate with a sample containing a ribosome inactivating protein to produce a product;
hybridizing said product to a template;
cleaving said product with an endonuclease;
extending said product by a polymerization reaction to form an oligonucleotide strand complementary to said template; and
detecting said oligonucleotide strand complementary to said template.

### Claim 2 (depends on 1)

The process of claim 1, wherein said endonuclease is apurinic/apyrimidinic endonuclease.

### Claim 3 (depends on 1)

The process of claim 1, wherein said substrate does not form a thermodynamically stable stem-loop.

### Claim 4 (depends on 1)

The process of claim 1, wherein said template is selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 6.

### Claim 5 (depends on 1)

The process of claim 1, wherein said detecting is by a method selected from the group consisting of RT-PCR, mass spectrometry, and HPLC.

### Claim 6 (depends on 1)

The process of claim 1, wherein said extending said product by a polymerisation reaction uses primers selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 8.

### Claim 7

A process for detecting ribosome inactivating protein in a sample, comprising:
providing an oligonucleotide substrate of SEQ ID NO: 1;
contacting said substrate with a sample containing a ribosome inactivating protein to produce a product;
hybridizing said product to a template of SEQ ID NO: 6;
cleaving said product with an endonuclease;
extending said product by a polymerization reaction to form an oligonucleotide strand complementary to said template; and
detecting said oligonucleotide strand complementary to said template by RT-PCR.

### Claim 8 (depends on 7)

The process of claim 7, wherein said endonuclease comprises an apurinic/apyrimidinic endonuclease.

### Claim 9 (depends on 7)

The process of claim 7, wherein detecting said oligonucleotide strand is by using a probe of SEQ ID NO: 7; wherein hybridizing said probe is under conditions suitable for a polymerase chain reaction; and detecting a first detection signal from said probe hybridized to said oligonucleotide strand complementary to said template.

### Claim 10 (depends on 9)

The process of claim 9, further comprising comparing said first detection signal to a second detection signal, wherein said second detection signal results from the hybridization of a probe complementary to a second template sequence.

### Claim 11 (depends on 10)

The process of claim 10, wherein said second detection signal is generated in parallel with said first detection signal.

### Claim 12 (depends on 11)

The process of claim 11, wherein said first detection signal is compared to a third detection signal from a ribosome inactivating protein calibrator extracted in parallel to said sample.

### Claim 13 (depends on 12)

The process of claim 12, wherein said ribosome inactivating protein calibrator comprises a known amount of ribosome inactivating protein and a known amount of a medium similar to the sample.

### Claim 14 (depends on 13)

The process of claim 13, further comprising confirming said detection signal with a protein based ribosome inactivating protein detection assay.

### Claim 15 (depends on 7)

The process of claim 7, wherein said detecting diagnoses ribosome inactivating protein intoxication in a human.

### Claim 16 (depends on 7)

The process of claim 7, wherein said oligonucleotide strand complementary to said template is generated by PCR extension from a product generated by a purified and titered ribosome inactivating protein solution.