Method for using polymyxin-coated substrate for lipopolysaccharide detection

• US 5,510,242 A
• Filed: 07/07/1993
• Issued: 04/23/1996
• Est. Priority Date: 05/18/1990
• Status: Expired due to Fees

• Current Assignee: Ricoh Kyosan Inc.
• Original Assignee: Burton W. Blais, Hiroshi Yamazaki
• Inventors: Yamazaki, Hiroshi, Blais, Burton W.
• Primary Examiner(s): Walsh, Stephen G.
• Assistant Examiner(s): KEMMERER, ELIZABETH
• Application Number:
  US08/087,013
• Time in Patent Office: 1,021 Days
• Field of Search: 435/2, 435/4, 435/7.21, 435/7.32, 435/69.6, 435/184, 435/28, 435/197, 435/7.92, 530/319, 436/518, 436/818, 436/543, 424/88, 424/92, 424/858
• US Class Current: 435/7.32
• CPC Class Codes: G01N 33/56911 Bacteria

Abstract

An improved device is provided which may be used, e.g. for immunoassay of lipopolysaccharides or for removing LPS pyrogens from aqueous solutions, or for removing LPS endotoxins from wounds. Such device comprises, in combination, a substrate, e.g. plastic, i.e. polystyrene, polycarbonate, polymethylmethacrylate or polyvinyl chloride, or a woven cloth, i.e. a rayon/polyester cloth or a polyester cloth, or a non-woven cloth, i.e. a rayon/polyester cloth, or a polyester cloth, or paper, which is adapted to receive a sample to be tested, and an oligopeptide, or a hydrophobic polypeptide or a polymyxin, e.g. polymyxin B, polymyxin B₁, polymyxin B₂, polymyxin D₁, polymyxin D₂, or polymyxin E, adhered to the substrate.

Claims

1. A process for the detection of lipopolysaccharide present on the cell walls of a target Gram negative bacteria which process comprises the steps of:

a) heating a sample of bacteria in a detergent solution to extract lipopolysaccharide antigens from said cell walls of said Gram negative bacteria;

b) contacting said solution with a device consisting essentially of in combination, a macroporous, hydrophobic material as a substrate, said substrate comprising a cloth composed of hydrophobic synthetic polymeric fibers, selected from the group consisting of polyester, polypropylene, and nylon and blends thereof with rayon which are either woven or non-woven into a physically-structurally-stable cloth of more than about 200 μ

m thickness, such that the pores exceed about 20 μ

m in diameter, said substrate being adapted to receive a sample to be tested, and, bound by simple adsorption to said substrate, a polymyxin;

c) washing said device; and

d) detecting the presence of said predetermined lipopolysaccharide by contacting an antibody indicator conjugate specifically binding said lipopolysaccharide to said device.

2. A process according to claim 1 wherein said detergent comprises a detergent selected from the group consisting of ethylenediaminetetracetic acid, sodium cholate, sodium deoxycholate, alkylaryl polyether alcohols, alkylaryl sulfonates, alkylaryl sulfates and sodium dodecyl sulfate, dissolved in a medium selected from the group consisting of phosphate buffered saline and a suitable liquid enrichment medium.

3. The process of claim 2 wherein said suitable liquid enrichment medium is selected from the group consisting of buffered peptone water, and a selective medium, and wherein said detergent is selected from the group consisting of ethylenediaminetetraacetic acid and sodium deoxycholate.

4. The process of claim 2 wherein said enrichment medium is selected from the group consisting of buffered peptone water with yeast extract, tetrathionate broth, selenite cysteine broth and nutrient broth.

5. A process according to claim 1 for the detection of bacterial lipopolysaccharide wherein said indicator conjugate is an antibody radioisotope complex suitable in radioimmunoassay.

6. A process according to claim 1 for the detection of bacterial lipopolysaccharide wherein said indicator conjugate is an antibody-enzyme complex adapted to cause a colour reaction with the addition of a chromogen suitable for enzyme immunoassay.

7. A process according to claim 1 for the detection of bacterial lipopolysaccharide wherein said indicator conjugate is a fluorescent label complex suitable for fluorescent immunoassay.

8. A process according to claim 1 for the detection of bacterial lipopolysaccharide wherein said indicator conjugate is a chemiluminescent label complex suitable for chemiluminescent immunoassay.

9. A process according to claim 1 wherein said polymyxin is selected from the group consisting of polymyxin B, polymyxin B₁, polymyxin B₂, polymyxin D₁, polymyxin D₂, and polymyxin E.

10. A process according to claim 1 wherein said antibody indicator conjugate to enable an assay of said lipopolysaccharide is selected from the group consisting of enzyme immunoassay, radioimmunoassay and fluorescent immunoassay.

11. A process according to claim 1 wherein said sample is a comminuted food sample;

wherein said substrate is a cloth selected from the group consisting of woven rayon/polyester cloth, woven polyester cloth, non-woven rayon/polyester cloth, and non-woven polyester cloth;

wherein said peptide is a polymyxin; and

wherein said heating step is an incubating step.

12. The process of claim 11 comprising the further steps of preparing a serial dilution series of said food sample and assaying said dilution series in enrichment medium.