# US 5,705,398 A · Methods for identifying inhibitors of LPS-mediated LBP binding

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- Number: US 5,705,398 A
- Document ID: US5705398A
- Status: Granted
- Kind: A
- Application number: 08/205,719
- Filed: 1994-03-02
- Granted: 1998-01-06
- Earliest claimed: 1994-03-02
- Assignees: The Scripps Research Institute
- Inventors: Douglas N. Mintz; Peter Tobias; Richard Ulevitch
- Web page: https://patentgrep.com/patent/US5705398A

## CPC

- G01N 33/56911: {Bacteria}
- G01N 33/566: using specific carrier or receptor proteins as ligand binding reagents {where possible specific carrier or receptor proteins are classified with their target compounds}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present invention describes a rapid method for the identification of compounds/reagents which inhibit lipopolysaccharide (LPS) binding to LPS binding protein (LBP), and kits for practicing the method.

## Claims

### Claim 1

A method for identifying a compound which inhibits lipopolysaccharide (LPS) binding to LPS binding protein (LBP), which method comprises:
a) measuring the fluorescence emitted by a first binding reaction admixture in solution that comprises:
i) fluoresceinated LPS, and
ii) isolated LBP;
b) measuring the fluorescence emitted by a second binding reaction admixture in solution that comprises:
i) fluoresceinated LPS,
ii) isolated LBP, and
iii) said compound; and
c) identifying a reduction in measured fluorescence emitted by said second binding reaction admixtures compared to said first binding reaction admixture, thereby identifying said inhibition activity of said compound.

### Claim 2 (depends on 1)

The method of claim 1 wherein said fluoresceinated LPS is fluorescein-re595 LPS.

### Claim 3 (depends on 1)

The method of claim 1 wherein said LBP is human or rabbit LBP.

### Claim 4 (depends on 1)

The method of claim 1 wherein said fluoresceinated LPS is present in said binding reaction admixture at a concentration of from about 1 to about 100 nanograms per milliliter.

### Claim 5 (depends on 1)

The method of claim 1 wherein said LBP is present in said binding reaction admixture at a concentration of from about 10 to about 1000 nanograms per milliliter.

### Claim 6

A method for identifying a compound which inhibits lipopolysaccharide binding protein (LBP)-dependent binding of lipopolysaccharide (LPS) to monocyte receptor CD14, which method comprises:
a) measuring the fluorescence emitted by a first binding reaction admixture in solution that comprises:
i) fluoresceinated LPS,
ii) isolated LBP, and
iii) isolated CD14;
b) measuring the fluorescence emitted by a second binding reaction admixture in solution that comprises:
i) fluoresceinated LPS,
ii) isolated LBP,
iii) isolated CD14, and
iv) said compound; and
c) identifying a reduction in measured fluorescence emitted by said second binding reaction admixture compared to said first binding reaction admixture, thereby identifying said inhibition activity of said compound.

### Claim 7 (depends on 6)

The method of claim 6 wherein said fluoresceinated LPS is fluorescein-re595 LPS.

### Claim 8 (depends on 6)

The method of claim 6 wherein said LBP is human or rabbit LBP.

### Claim 9 (depends on 6)

The method of claim 6 wherein said isolated CD14 has an amino acid residue sequence shown in SEQ ID NO 2.

### Claim 10 (depends on 6)

The method of claim 6 wherein said fluoresceinated LPS is present in said binding reaction admixture at a concentration of from about 1 to about 100 nanograms per milliliter.

### Claim 11 (depends on 6)

The method of claim 6 wherein said LBP is present in said binding reaction admixture at a concentration of from about 10 to about 1000 nanograms per milliliter.

### Claim 12 (depends on 6)

The method of claim 6 wherein said isolated CD14 is present in said binding reaction admixture at a concentration of from about 5 to 30 micrograms per milliliter.