Use of porphyrins as a universal label

• US 6,001,573 A
• Filed: 10/23/1997
• Issued: 12/14/1999
• Est. Priority Date: 06/14/1996
• Status: Expired due to Term

• Current Assignee: Packard Bioscience B.V.
• Original Assignee: Packard Bioscience B.V.
• Inventors: Roelant, Chris
• Primary Examiner(s): Houtteman, Scott W.
• Application Number:
  US08/956,856
• Time in Patent Office: 782 Days
• Field of Search: 455/6, 530/350
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72

Abstract

A process is provided for using porphyrin or porphyrin derived compounds as universal labels for various assays and other quantification techniques without the need for a bridging agent to couple the label to the target particles. Particles which can be labeled include beads, microorganisms, cells, and molecules. The porphyrin label irreversibly attaches to target particles and afterwards can be detected and quantified by any number of ways, such as chemiluminometrically, fluorometrically or radiometrically in an amount which is proportional to the number of labeled particles.

Claims

1. A process for detecting particles comprising the steps of:

a) mixing porphyrin, in the absence of a bridging agent, with said particles for a time sufficient for the porphyrin to bind to the particles;

b) separating the porphyrin labeled particles from the unbound porphyrin; and

c) detecting said porphyrin labeled particles.

2. The process according to claim 1, wherein said porphyrin labeled particles are detected by chemiluminometric, radiometric, or fluorometric processes.

3. The process according to claim 1, wherein said porphyrin is a protoporphyrin IX.

4. The process according to claim 1, wherein said porphyrin is a ferroprotoporphyrin.

5. The process according to claim 4, wherein said ferriprotoporphyrin is hematin or hemin.

6. The process according to claim 1, wherein the quantification of the porphyrin labeled particles is carried out by chemiluminometric processes and wherein a luminescent probe and an oxidizer are admixed with the separated porphyrin labeled particles.

7. The process according to claim 6, wherein said luminescent probe is a 2,3-dihydro-1,4-phthalazinedione.

8. The process according to claim 6, wherein said oxidizer is selected from the group consisting of perborate, hydrogen peroxide, hydroperoxide, endoperoxide, and an oxidizer producing enzyme.

9. The process according to claim 6, wherein the chemiluminometric process is additionally conducted in the presence of a chelator and a buffer to maintain pH.

10. The process according to claim 9, wherein the chelator is EDTA or desferrioxamine.

11. The process according to claim 9, wherein the buffer is a borate, carbonate, tris(hydroxymethyl) aminomethane, or phosphate buffer.

12. The process according to claim 1, wherein said porphyrin is an isotopically labeled porphyrin and detection of the porphyrin labeled particles is carried out by radiometric processes.

13. The process according to claim 12, wherein said porphyrin is isotopically labeled with an atom selected from the group consisting of carbon-14, chlorine-36, cobalt-(57, 58, 60), gadolinium-153, iron-(55, 59), nickel-63, tritium, iodine-125, tin-113, zinc-65, phosphorus-(32, 33).

14. The process according to claim 1, wherein detection is conducted by radiometric processes in which SPA beads are combined with porphyrin labeled with iodine-125.

15. The process according to claim 1, wherein said porphyrin labeled particles are detected by virtue of the fluorescence of the porphyrin label.

16. The process according to claim 1, wherein the amount of porphyrin used for labeling is from about 10³ M to about 10⁵ M.

17. The process according to claim 1 wherein the particle to be detected is a molecule, bead, microorganism or cell.

18. An adhesion assay comprising the steps of:

a) providing a suspension of particles to be tested;

b) mixing porphyrin, in the absence of a bridging agent, with said suspension;

c) separating porphyrin labeled particles from unbound porphyrin;

d) incubating the porphyrin labeled particles with a target surface;

e) removing non-adherent porphyrin labeled particles; and

f) detecting the adherent porphyrin labeled particles.

19. The adhesion assay of claim 18, wherein the porphyrin labeled particles are separated from unbound porphyrin by centrifugation, magnetic separation, or filtration.

20. The adhesion assay of claim 18, wherein the adherent porphyrin labeled particles are detected by radiometric, fluorometric, or chemiluminometric processes.

21. A particle diameter or surface size assay comprising the steps of:

a) providing a suspension of a predetermined number of particles to be tested;

b) mixing porphyrin, in the absence of a bridging agent, with the suspension;

c) removing porphyrin labeled particles from unbound porphyrin;

d) detecting a signal generated by the predetermined number of particles; and

e) calculating the signal per particle as an indication of the diameter or surface size of the particles.

22. The particle diameter or surface size assay of claim 21, wherein the porphyrin labeled particles are removed from unbound porphyrin by centrifugation, magnetic separation, or filtration.

23. The particle diameter or surface size assay of claim 21, wherein the signal generated by the predetermined number of particles is detected by radiometric, fluorometric, or chemiluminometric processes.

24. A particle uptake study comprising the steps of:

a) providing a suspension of particles to be studied;

b) mixing porphyrin, in the absence of a bridging agent, with the suspension;

c) removing unbound porphyrin from porphyrin labeled particles;

d) resuspending labeled particles in an appropriate medium;

e) injecting the labeled particles and appropriate medium into biologic objects; and

f) tracing the injected particles.

25. The particle uptake study of claim 24, wherein the unbound porphyrin is removed from the porphyrin labeled particles by centrifugation, magnetic separation, or filtration.

26. The particle uptake study of claim 24, wherein the injected particles are traced by radiometric, fluorometric, or chemiluminometric processes.

27. An assay kit for detecting particles, in the absence of a bridging agent, comprising a first container containing porphyrin and a set of directions for chemiluminometric, radiometric, or fluorometric processes.

28. The assay kit according to claim 27, wherein the porphyrin is present in sufficient quantity to conduct multiple detection assays.

29. The assay kit according to claim 27, further comprising a second container containing a stabilized mixture of luminescence precursor and oxidizer.

30. The assay kit according to claim 29, wherein the second container further contains a buffer substance and a chelator.

31. The assay kit according to claim 27, wherein the porphyrin is isotopically labeled with a beta emitter and wherein the assay kit further comprises a second container containing a scintillation cocktail.

32. A composition comprising a porphyrin bound particle, wherein there is no bridging agent to couple the porphyrin to the particle.

33. The composition of claim 32, wherein the particle is a drug, drug metabolite, hormone, peptide, nucleotide, neurotransmitter, cholesterol, growth factor, oligonucleotide, antibody, antigen-binding fragment, serum protein, enzyme, polynucleotide, intracellular organelle, cell surface antigen, avidin, biotin, binding protein, nucleic acid, membrane probe, or nucleic acid probe.