Flow immunosensor method and apparatus

• US 5,183,740 A
• Filed: 02/23/1990
• Issued: 02/02/1993
• Est. Priority Date: 02/23/1990
• Status: Expired due to Term

• Current Assignee: the united states of america as represented by the secretary of the navy
• Original Assignee: the united states of america as represented by the secretary of the navy
• Inventors: Gaber Bruce P., Ligler, Frances S., Kusterbeck, Anne W., Wemhoff, Gregory A.
• Primary Examiner(s): Kepplinger, Esther L.
• Assistant Examiner(s): Spiegel, Carol A.
• Application Number:
  US07/486,024
• Time in Patent Office: 1,075 Days
• Field of Search: 435/7.36, 435/7.1, 435/7.92, 435/7.93, 435/805, 435/968, 435/961, 435/967, 435/7.32, 435/7.33, 435/7.34, 436/518, 436/529, 436/538, 436/541, 436/800, 436/801, 436/804, 422/70, 422/82, 422/82.01-82.09
• US Class Current: 435/7.32
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: Y10S 435/80 Elimination or reduction of contamination by undersired ferments, e.g. aseptic cultivation
• CPC Class Codes: Y10S 435/804 Single cell protein
• 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

Abstract

A method of detecting a target moiety comprising the steps of (a) providing an antibody specific to the target, (b) saturating the binding sites of the antibody with a labelled form of the target, (c) flowing a liquid containing the target past the saturated antibody, thereby (d) allowing the target to displace the labelled antigen, and (e) detecting the displaced labelled antigen with a detector for the label.

Also, the invention is accomplished by a flow immunosensor comprising: a liquid flowing through the immunosensor for moving a sample through the immunosensor; a receiving means for introducing the sample to the liquid stream; a flow control means for moving the liquid stream through the immunosensor; an exchanger connected to the sample receiving means in which the sample is brought in contact with labelled antigens of the target and whereby any target present is allowed to displace the labelled antigen; a detection apparatus connected to the exchanger for detecting any labelled antigen which is released; and a disposal means connected to the detection apparatus for disposing of or collecting of waste coming from the detection apparatus.

Claims

1. A method of detecting a target comprising,

(a) providing a solid phase-immobilized antibody capable of specifically binding to the target,

(b) exposing an antigen binding site of the antibody to a labelled analog of the target to form a solid phase-immobilized, labelled antigen-antibody complex,

(c) continuously flowing an aqueous medium suspected of containing the target past the solid phase-immobilized, labelled antigen-antibody complex at a flow rate allowing the target to displace the labelled analog from the labelled antigen-antibody complex under nonequilibrium conditions, and

(d) detecting the displaced labelled analog, the amount of said displaced labelled analog being directly proportional to the concentration of said target in said medium.

2. The method according to claim 1 wherein the target is selected from the group consisting of thyroxine;

high density lipoprotein;

low density lipoprotein;

cholesterol;

albumin;

alpha-1 antitrypsin;

beta-2 microglobulin;

ceruloplasmin;

C1 inhibitor;

C1q;

C-reactive protein;

cryfibrinogen;

ferritin;

myelin basic protein;

transferrin;

insulin;

human chorionic gonadotropin;

estrogen;

progesterone;

antigenic protein fragments from H. influenza, N. meningitides, S. pneumonia, Bordetella pertussis, Borrelia burgdorferi, pathogenic staphylococcus or pathogenic streptococcus;

thephylline;

digoxin;

L-dopa;

insulin;

cocaine and its metabolites;

cannabis metabolites;

heroin;

morphine metabolites;

trinitrotoluene;

cyclonite;

pentaerythritol tetranitrate;

nitrobenzene; and

isothiocyanates.

3. The method according to claim 1 wherein the labelled antigen-antibody complex is soluble in said aqueous medium, and has one site per molecule at which the antibody of said complex is bound to the labelled antigen of said complex.

4. The method according to claim 3 wherein the label of the labelled complex is selected from the group consisting of radiolabels, fluorophores, chromophores, and electron spin labels.

5. The method according to claim 4 wherein the label of the labelled complex is selected from the group consisting of radiolabels and fluorophores.

6. The method according to claim 1 wherein the aqueous medium is an aqueous buffer.

7. The method according to claim 6 wherein the buffer is selected from the group consisting of phosphate-buffered saline, borate-buffered saline, TRIS-saline, Alseiver'"'"'s solution and Ringer'"'"'s solution.

8. The method according to claim 1 wherein the aqueous medium flows at a rate of about 0.1 to 2.0 milliliters per minute.

9. The method according to claim 3 wherein the aqueous medium flows at a rate of about 0.1 to 2.0 milliliters per minute.

10. The method according to claim 6 wherein the aqueous medium flows at a rate of about 0.1 to 2.0 milliliters per minute.

11. The method according to claim 10 wherein the aqueous medium flows at a rate of about 0.3 to 0.8 milliliters per minute.

12. The method of claim 1, wherein said solid phase is a flow-through bed.