Labeled liposome particle compositions and immunoassays therewith

• US 4,193,983 A
• Filed: 05/16/1978
• Issued: 03/18/1980
• Est. Priority Date: 05/16/1978
• Status: Expired due to Term

• Current Assignee: DADE Behring Beteiligungs GmbH (Siemens AG)
• Original Assignee: Syva Co.
• Inventors: Ullman, Edwin F., Brinkley, John M.
• Primary Examiner(s): Fagelson, Anna P.
• Application Number:
  US05/906,514
• Time in Patent Office: 672 Days
• Field of Search: 424/1, 424/3, 424/7, 424/8, 424/11, 424/12, 424/13, 424/38, 260/112 R, 260/112 B, 23/230 B, 195/103.5 A, 195/103.5 L, 195/63
• US Class Current: 436/528
• CPC Class Codes: A61K 9/127 Liposomes
• CPC Class Codes: A61K 9/1271 Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers liposome as conjugate A61K47/6911
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: Y10S 435/961 including a step of forming, releasing, or exposing the antigen or forming the hapten-immunogenic carrier complex or the antigen per se
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 436/815 Test for named compound or class of compounds
• CPC Class Codes: Y10S 436/819 Multifunctional antigen or antibody
• CPC Class Codes: Y10S 436/829 Liposomes, e.g. encapsulation

Abstract

The subject invention concerns novel compositions for use in immunoassays, as well as immunoassays employing such novel compositions. The compositions comprise discrete charged colloidal particles comprised of small molecules which particles are capable of retaining their discrete character in an aqueous medium and composed of aggregates of lipophilic and/or amphiphilic organic molecules to which are bound non-covalently a label capable of producing a detectible signal and a ligand or an analog of the ligand capable of competing with a ligand for a ligand receptor. The discrete colloidal particle serves as a hub or nucleus for retaining the ligand or its analog and the label within a limited locus.

The compositions are prepared by individually covalently bonding the ligand and the label, when not naturally lipophilic, to a lipophilic (includes amphiphilic) compound, normally a phospholipid. Depending upon the nature of the particle, the amphiphilic conjugated ligand and label are combined with the particle or alternatively may be combined with the compounds employed for preparing the particle under particle forming conditions. Particles are then obtained having the analog of the ligand and the label bound to the particle.

The compositions find use in immunoassays where an interaction between the label and receptor provides a means for modulating a detectible signal. The interaction can be as a result of quenching or modification of fluorescence, where the label is a fluorescer, steric inhibition of the approach of a signal modifier to the label, such as a label receptor or with an enzyme label, an antienzyme or enzyme inhibitor, the inhibition of cleavage of an enzyme labile bond or the cooperative interaction of two labels, such as two enzymes, where the product of one enzyme is a substrate of another enzyme.

Claims

1. In an immunoassay for the determination of an analyte, where the analyte is a member of a specific binding pair consisting of ligand and antiligand and wherein a label is employed which provides a detectible signal, the value of which is affected by the proximity of said antiligand to said label due to the presence of the bulk of said antiligand or a compound conjugated to said antiligand which interacts with said label,

wherein said immunoassay comprises combining in an aqueous medium:

(1) a sample suspected of containing said analyte, (2) a reacgent comprising ligand or ligand analog and label, wherein said ligand analog is capable of specific binding to said antiligand, and (3) antiligand, when ligand is the analyte or said antiligand is conjugated with said compound which interacts with said label; and

determining the value of said detectible signal as compared to said value determined with a sample having a known amount of analyte;

the improvement which comprises employing as said reagent discrete colloidal particles comprised of a major portion of lipophilic compounds and a minor portion of label and ligand or ligand analog, wherein said label is covalently conjugated to at least one of said lipophilic compounds, and the ligand or ligand analog are lipophilic or made so by conjunction to a lipophilic compound.

2. A method according to claim 1, wherein said lipophilic compound is amphiphilic.

3. A method according to claim 2, wherein said amphiphilic compound is a phosphatidyl compound.

4. A method according to claim 1, wherein said label is a fluorescent compound.

5. A method according to claim 4, wherein said fluorescent compound is conjugated to a phosphatidyl group.

6. A method according to claim 5, wherein said ligand is cardiolipin and said analyte is anticardiolipin.

7. An immunoassay for the determination of an analyte, wherein the analyte is a member of a specific binding pair consisting of ligand and antiligand and wherein a label is employed which provides a detectible signal, the value of which is affected by the proximity of said antiligand to said label due to the presence of the bulk of said antiligand or a compound conjugated to said antiligand which interacts with said label, and

wherein a reagent is employed having both label and ligand or a ligand analog, where ligand analog is capable of specifically binding to said antiligand, said reagent comprising a liposome of amphiphilic molecules having from about 0.05 to 15 mole percent of ligand or ligand analog conjugated to an amphiphile and from 0.05 to 15 mole percent of a label conjugated to at least one of said amphiphile molecules;

wherein said immunoassay comprises combining in an aqueous medium at a pH in the range of about 5 to 10 and at a temperature in the range of about 10°

to 50°

C.;

a sample suspected of containing said analyte;

said reagent;

and antiligand, when ligand is the analyte or said antiligand is conjugated with a compound which interacts with said label; and

determining the value of said detectible signal as compared to said value determined with a sample having a known amount of analyte.

8. A method according to claim 7, wherein said label is a fluorescer and antifluorescer is added to said medium.

9. A method according to claim 8, wherein said fluorescer is a fluorescein derivative.

10. A method according to any of claims 7 to 9, wherein said ligand is cardiolipin.

11. A method according to claim 7, where from about 16 to 99.5 mole percent of said liposome is phospholipid.

12. A method according to claim 11, where up to 40 mole percent is a cholestanol derivative.

13. An immunoassay for the determination of the presence of anticardiolipin in human serum which comprises:

combining in an aqueous buffered medium at a pH in the range of about 6.5 to 9.5;

(1) a human serum sample suspected of containing anticardiolipin

(2) liposomes comprised of from about 60 to 99.5 mole percent of a phosphatidyl compound, from about 0.05 to 5 mole percent of cardiolipin and from about 0.1 to 10 mole percent of a phosphatidyl-fluorescein conjugate; and

(3) antifluorescein; and

determining the amount of fluorescence from said assay medium as compared to the amount of fluorescence obtained from an assay medium having a known amount of anticardiolipin.

14. An immunoassay composition of matter comprising a liposome having from 16 to 99.5 mole percent of phospholipid, up to 40 mole percent of cholestanol, from about 0.05 to 15 mole percent of a fluorescent label bonded to a phospholipid, wherein said fluorescent label has an extinction coefficient of at least about 10⁴ or at above 350 nm and a hapten of from about 125 to 2,000 molecular weight bonded to at least one phospholipid.

15. An immunoassay composition of matter according to claim 14, wherein said fluorescent label is a fluorescein derivative.

16. An immunoassay composition of matter having at least about 16 mole percent of phospholipid, up to 40 mole percent of a cholestanol, from 0.05 to 15 mole percent of a fluorescent label bonded to a phospholipid and from about 0.05 to 15 mole percent of cardiolipin.

17. An immunoassay composition of matter comprising a liposome having from about 60 to 99.5 mole percent of a phospholipid, from about 0.05 to 5 mole percent of cardiolipin, and from about 0.1 to 10 mole percent of phosphatidyl-fluorescer conjugate.