Methods for cellular or microorganism capture and quantification using bioluminescence regenerative cycle (BRC) assays

• US 20050112601A1
• Filed: 02/27/2004
• Published: 05/26/2005
• Est. Priority Date: 02/27/2003
• Status: Abandoned Application

• Current Assignee: Xagros Technologies, Inc.
• Original Assignee: Xagros Technologies, Inc.
• Inventors: Hassibi, Arjang, Ghazvini, Siavash
• Application Number:
  US10/788,579
• Publication Number:
  US 20050112601A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence

Abstract

The methods, apparatus and compositions disclosed herein concern the detection, identification and/or quantification of target cells and/or microorganisms in samples. The assays are based on light emission detected from a bioluminescence regenerative cycle (BRC). Light emission may be related to cell and/or microorganism number through the number of ATP and PPi molecules per cell or microorganism. In certain embodiments of the invention, specific target cells and/or microorganisms may be separated from samples using one or more capture molecules, such as antibodies. The cells and/or microorganisms may be lysed, the contents purified in whole or in part and the ATP and PPi contents determined by BRC. Other embodiments of the invention concern apparatus comprising a series of chambers connected by a monodirectional flow channel, each chamber comprising an affinity matrix with one or more binding moieties attached. In certain embodiments, a multiplex assay may be performed using both antibodies and oligonucleotide probes specific for a pathogen of interest.

Claims

1. ) A method comprising:

a) obtaining one or more samples;

b) obtaining one or more types of target cells and/or microorganisms from each sample;

c) performing a bioluminescence regenerative cycle (BRC) assay to detect ATP and inorganic phosphate (PPi) in the cells and/or microorganisms; and

d) estimating the number of target cells and/or microorganisms from the BRC assay.

2. ) The method of claim 1, further comprising using one or more binding moieties to separate one or more types of target cells and/or microorganisms from the sample.

3. ) The method of claim 2, wherein the capture moiety is an antibody.

4. ) The method of claim 3, wherein the antibody is attached to a magnetic bead, glass bead, plastic bead, nitrocellulose membrane, nylon membrane, chromatography support or other solid surface.

5. ) The method of claim 1, further comprising lysing the cells and/or microorganisms.

6. ) The method of claim 5, further comprising partially purifying the ATP and PPi from the lysed cells and/or microorganisms.

7. ) The method of claim 1, wherein the sample is blood, serum, plasma, cerebrospinal fluid, lymphatic fluid, urine, stool, semen, lacrimal fluid, saliva, sputum, a biopsy sample, a tissue scraping, a swab sample, an endoscopic sample, a cell sample, a tissue sample, a food sample, a water sample, an environmental sample or an air sample.

8. ) The method of claim 1, further comprising accumulating the photon count over a time interval.

9. ) The method of claim 1, further comprising adding adenylate kinase and AMP substrate or pyruvate kinase and phosphoenolpyruvate to the BRC assay.

10. ) The method of claim 1, further comprising adding between 0.01 and 10 attomole of purified pyrophosphate or ATP to each assay to reduce background light emission.

11. ) A kit for performing BRC assays comprising:

a) one or more BRC reagents; and

b) a standard solution.

12. ) The kit of claim 11, wherein the standard solution comprises a known amount of ATP and/or PPi.

13. ) The kit of claim 11, wherein the BRC reagents comprise at least one reagent selected from the group consisting of luciferin, luciferase, ATP sulfurylase, adenosine 5′

-phosphosulphate and BRC buffer.

14. ) An apparatus comprising:

a) a light tight slide;

b) a plurality of chambers in the slide, the chambers connected by a mono-directional flow channel;

c) an inlet and an outlet connected to the flow channel; and

d) a sealed secondary inlet connected to each chamber.

15. ) The apparatus of claim 14, further comprising an affinity matrix in each chamber.

16. ) The apparatus of claim 15, wherein each affinity matrix is attached to at least one binding moiety.

17. ) The apparatus of claim 16, wherein the binding moieties are antibodies or capture oligonucleotides.

18. ) The apparatus of claim 17, wherein each chamber comprises at least one antibody and at least one capture oligonucleotide, both specific for the same pathogen.