PROCESS FOR ASSAYING FOR BIOLOGICALLY ACTIVE MOLECULES

• US 3,850,578 A
• Filed: 02/19/1974
• Issued: 11/26/1974
• Est. Priority Date: 03/12/1973
• Status: Expired due to Term

• Current Assignee: Harden M. Mcconnell
• Original Assignee: Harden M. Mcconnell
• Inventors: McConnell, Harden M.
• Primary Examiner(s): Wolk, Morris O.
• Assistant Examiner(s): Hagan, Timothy W.
• Application Number:
  US05/443,550
• Time in Patent Office: 280 Days
• Field of Search: 23/23B 424/12
• US Class Current: 436/536
• CPC Class Codes: G01N 33/586 Liposomes, microcapsules or cells
• CPC Class Codes: G01R 33/281 Means for the use of in vitro contrast agents G01R33/282 takes precedence; involving use of a contrast agent in MR imaging G01R33/5601; in vivo contrast agents A61K49/0002
• CPC Class Codes: Y10S 436/803 Stabe free radicals, e.g. spin immunoassay
• CPC Class Codes: Y10S 436/829 Liposomes, e.g. encapsulation

Abstract

Immunoassay technique employing sacs having a lysable membrane and at least one determinant or epitopic site recognizable by an antibody. Enclosed in the sacs are water soluble stable free radical compounds, which are capable of escape from the sac upon lysis. The immunoassay can be employed for the determination of an epitope containing compound, antibodies or complement. Two basic modes are employed. The first mode depends upon the difference in the EPR spectrum of the stable free radical when enclosed in the sac, at relatively high localized concentration, and when free in solution, at relatively low concentration. The amount of the free radical compound free in solution is dependent upon available antibody bound to the sacs and available complement. The second mode can be used for the determination of an epitope containing compound or antibody. In this mode, the antibody agglutinates the sacs, the agglutinated sacs are separated from the non-agglutinated sacs and either or both of the differentiated sacs lysed and the amount of free radical determined. The immunoassay is extremely sensitive and has a high multiplication factor, because a single event results in the freeing of a large number of free radical molecules.

Claims

1. A METHOD FOR ASSAYING FOR AN ORGANIC COMPOSITION WHICH IS A COMPOUND HAVING AT LEAST ONE EPITOPIC SITE, AN ANTIBODY CAPABLE OF SPECIFICALLY BINDING TO A SPECIFIC EIPOTOPIC SITE OR COMPLEMENT IN AN UNKNOWN SUSPECTED OF CONTAINING ONE OF SAID ORGAINC COMPOSITION WHICH COMPRISES:

COMBINING IN AN AQUEOUS BUFFERED MEDIUM UNDER COMPLEMENT LYSING CONDITIONS;

2. sacs enclosing an aqueous solution, substantially isotonic with said medium, of stable free radicals at a concentration sufficient to provide peak broadening of the EPR spectrum of said free radical, and having at least one epitopic site at the surface of said sacs;

3. ANTIBODY CAPABLE OF BINDING TO SAID EPITOPIC SITES OF SAID SAC;

AND

4. complement;

with the proviso that the amount of antibody or complement in said medium is known in the absence of said unknown, when antibody or complement respectively are being detected in said unknown and at least one of said epitopic sites of said sacs and said compound are the same, when said compound is being determined;

whereby lysis of said sac occurs upon the binding of antibody and complement to said sac; and

determining at least one point of the EPR spectrum of said medium as compared to the EPR spectrum of said medium at a known concentration of said organic composition.

5. A method according to claim 4, wherein said sacs are passively sensitized with a compound having at least one epitopic site.

6. A method according to claim 4, wherein said sacs are actively sensitized by a compound having at least one epitopic site.

7. A method for assaying an unknown suspected of containing a compound having at least one epitopic site which comprises:

combining said unknown and antibodies capable of binding to said epitopic site to provide an unknown-antibody aqueous solution;

combining said unknown-antibody solution with sacs to provide an aqueous buffered medium, wherein said sacs enclose an aqueous solution, substantially isotonic with said medium, of stable free radicals at a concentration sufficient to provide peak broadening of the EPR spectrum of said free radicals and having at least one epitopic site at the surface of said sacs recognized by said antibody, and complement, under lysing conditions, whereby lysis of said sacs occurs upon the binding of antibody and complement to said sacs; and

determining at least one point of said EPR spectrum of said medium as compared to a medium assayed under the same conditions having a known amount of said compound.

8. A method according to claim 7, wherein said compound is an antigen.

9. A method for assaying an unknown suspected of containing an antibody which comprises:

combining in an aqueous buffered medium under complement lysing conditions;

10. A method for assaying an unknown suspected of containing complement which comprises:

combining in an aqueous buffered medium under complement lysing conditions;

11. A method for assaying an unknown suspected of containing a compound having at least one epitopic site which comprises:

combining in an aqueous buffered medium said unknown with sacs enclosing an aqueous solution, substantially isotonic with said medium, of stable free radicals at a concentration sufficient to provide peak broadening of the EPR spectrum of said free radicals, and having a plurality of epitopic sites at the surface of said sac capable of binding to the same antibody to which said compound binds; and

antibody for said epitopic site, wherein the proportion of sacs and antibody results in agglutination of a substantial proportion of said sacs and antibody in the absence of said unknown;

separating agglutinated sacs from said medium;

lysing the sacs in at least one of said agglutinated sacs or said separated medium; and

determining at least one point of the EPR spectrum of said lysed agglutinated sacs or said lysed sacs in said medium as compared to the EPR spectrum of the lysed sacs obtained with a known concentration of said compound.