Phage particle diagnostic reagents

Patent metadata

Field	Value
Publication number	US-9658224-B2
Application number	US-8561806-A
Country	US
Kind code	B2
Filing date	Nov 14, 2006
Priority date	Nov 29, 2005
Publication date	May 23, 2017
Grant date	May 23, 2017

Abstract

The present invention relates to novel methods for detecting a member of a known binding pair in a sample, including a cell, where one member of the pair (termed the “receptor”) is expressed by a bacteriophage, which phage is then used to detect the presence of the other member of the pair (termed the “ligand” or “target”). Rather than detecting the binding of the phage using antibody-based technology, the present invention relates to detecting marker molecule associated with the phage. In one aspect, the invention relates to identifying an antigen-bearing moiety (e.g., a red blood cell antigen) of interest present on a cell, e.g., a red blood cell, using antibody-displaying bacteriophage, as well as detecting anti-red blood cell auto- or alloantibodies and/or complement in a sample, using antiglobulin reagent-displaying bacteriophage and detecting a marker molecule associated with the phage. In one aspect, the phenotype of the phage is not linked with the genotype of the phage.

First claim

What is claimed:

1. A method of generating an antibody-displaying bacteriophage that does not contain a nucleic acid sequence encoding the displayed antibody, the method comprising the steps of:
a) administering a non-phage vector comprising a nucleic acid that lacks a phage packaging sequence to a bacterial cell, wherein the nucleic acid encodes a fusion protein comprising an antibody and a polypeptide that will be displayed on the outer surface of a bacteriophage, and wherein the bacterial cell produces the fusion protein;
b) infecting the bacterial cell with a phage, wherein the phage comprises a detectable marker molecule, wherein phage particles are produced in the bacterial cell; and
c) assembling a bacteriophage in the bacterial cell, wherein the bacteriophage comprises the detectable marker molecule and displays the fusion protein on its outer surface and does not contain the nucleic acid sequence encoding the displayed antibody.

2. The method of claim 1 , wherein the marker molecule is selected from the group consisting of a marker nucleic acid, a fluorescent molecule, a polypeptide, a lipid, a carbohydrate, a ligand, a receptor, an enzyme, a substrate, and an inorganic molecule.

3. The method of claim 1 , wherein the antibody binds specifically to a red blood cell antigen.

4. The method of claim 3 , wherein the red blood cell antigen is selected from the group consisting of A, B, Rh(D), Rh(C), Rh(c), Rh(E), Rh(e), K, k, Js a , Js b , Kp a , Le a , Le b , Lu a , Lu b , Fy a , Fy b , M, N, S, s, Do a , Do b , Jk a , and Jk b .

5. The method of claim 1 , wherein the antibody binds specifically to an antigen selected from the group consisting of a lymphocyte antigen, a monocyte antigen, and a granulocyte antigen.

6. The method of claim 1 , wherein antibody binds specifically to a platelet antigen.

7. The method of claim 6 , wherein the platelet antigen is selected from the group consisting of HPA-1a, HPA-1b, HPA-2a, HPA-2b, HPA-3a, HPA-3b, HPA-4a, HPA-4b, HPA-5a, HPA-5b, HPA-6b, HPA-7b, HPA-8b, HPA-9b, HPA-10b, Gov a , and Gov b .

8. The method of claim 1 , wherein the marker molecule comprises a nucleic acid sequence complementary to a molecular beacon probe.

9. The method of claim 8 , wherein the molecular beacon probe comprises a fluorophore.

10. The method of claim 2 , wherein the marker molecule is a marker nucleic acid.

11. A kit for detecting the presence of an antigen-bearing moiety on a cell, the kit comprising the bacteriophage generated according to the method of claim 1 ;
the kit further comprising an applicator and an instructional material for the use thereof.

12. A method of detecting the presence of an antigen-bearing moiety on a cell, the method comprising the steps of:
a) generating an antibody-displaying bacteriophage according to the method of claim 1 ;
b) contacting a cell with the bacteriophage;
c) denaturing any bacteriophage specifically bound with the cell to release the marker molecules; and
d) detecting the marker molecules, wherein detecting the marker molecule detects the presence of the antigen-bearing moiety on the cell.

13. A method of detecting the presence of at least two different antigen-bearing moieties on a cell, the method comprising the steps of:
a) generating a first antibody-displaying bacteriophage with a first marker molecule according to the method of claim 1 ;
b) generating a second antibody-displaying bacteriophage with a second marker molecule according to the method of claim 1 ;
c) contacting the cell with the first bacteriophage;
d) contacting the cell with the second bacteriophage;
e) detecting the binding of the first bacteriophage with the first antigen-bearing moiety by detecting the presence of the first marker molecule, wherein detecting the first marker molecule detects the presence of the first antigen-bearing moiety on the cell; and
f) detecting the binding of the second bacteriophage with the second antigen-bearing moiety by detecting the presence of the second marker molecule, wherein detecting the second marker molecule detects the presence of the second antigen-bearing moiety on the cell.

14. The method of claim 13 , wherein the first marker molecule is a first marker nucleic acid, and further wherein the second marker molecule is a second marker nucleic acid.

15. The method of claim 14 , wherein the first and second marker nucleic acids are detected by assaying the melting temperatures of the first and the second marker nucleic acids.

16. A method of detecting the presence of an anti-red blood cell antibody in human serum, the method comprising the steps of:
a) contacting a human red blood cell expressing at least one human red blood cell antigen on the surface of the cell with the serum;
b) washing the cell to remove any antibody bound non-specifically with the cell;
c) generating an anti-human globulin reagent-displaying bacteriophage according to the method of claim 1 , wherein the antibody is the anti-human globulin reagent;
d) contacting the cell with the bacteriophage;
e) washing the cell to remove any bacteriophage bound non-specifically with the cell;
f) denaturing the bacteriophage specifically bound with the cell to release the marker molecule; and
g) detecting the marker molecule, wherein detecting the marker molecule detects the presence of the anti-red blood cell antibody in the serum.

17. A method of detecting the presence of an anti-red blood cell antibody in a human, the method comprising the steps of:
a) obtaining a red blood cell from the human;
b) washing the cell to remove any antibody bound non-specifically with the cell;
c) generating an anti-human globulin reagent-displaying bacteriophage according to the method of claim 1 , wherein the antibody is the anti-human globulin reagent;
d) contacting the cell with the bacteriophage;
e) denaturing the bacteriophage specifically bound with the cell to release the marker molecule; and
f) detecting the marker molecule, wherein detecting the marker molecule detects the presence of the anti-red blood cell antibody in the human.

18. A method of detecting the presence of an antigen-bearing moiety in a composition, the method comprising the steps of:
a) generating an antibody-displaying bacteriophage according to the method of claim 1 ;
b) contacting the composition with the bacteriophage; and
c) detecting the marker molecule, wherein detecting the marker molecule detects the presence of the antigen-bearing moiety on the cell.

19. The method of claim 12 , further comprising amplifying the marker molecule prior to step (d).

20. The method of claim 12 , the method further comprising washing the cell between step (b) and step (c).

21. The method of claim 19 , wherein the marker molecule is amplified using polymerase chain reaction (PCR).

22. The method of claim 19 , wherein the marker molecule is amplified by transcription using immuno-detection amplified by T7 RNA (DAT).

23. The method of claim 19 , wherein the marker nucleic acid is detected by assaying the melting temperature of the marker nucleic acid.

Assignees

• Siegel Donald
• Barbas Iii Carlos F
• Univ Pennsylvania
• The Scripps Res Insitute

Inventors

• Siegel Donald
• Barbas Iii Carlos F

Classifications

• G01N33/6854

  Immunoglobulins · CPC title

• G01N33/555 Primary

  Red blood cell · CPC title