Multiepitopic immunometric assay

• US 5,011,771 A
• Filed: 09/24/1987
• Issued: 04/30/1991
• Est. Priority Date: 04/12/1984
• Status: Expired due to Fees

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Wands, Jack R., Bellet, Dominique
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Sisson, Bradley L.
• Application Number:
  US07/102,766
• Time in Patent Office: 1,314 Days
• Field of Search: 435/7, 435/240.27, 435/810, 436/531, 436/535, 436/548, 436/800, 436/804, 436/808, 436/819, 436/813-815
• US Class Current: 435/7.94
• CPC Class Codes: G01N 2470/04 Sandwich assay format
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/57476 involving oncofetal proteins
• CPC Class Codes: G01N 33/577 involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens; G01N33/53 - G01N33/576 take precedence
• CPC Class Codes: G01N 33/76 Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/804 Radioisotope, e.g. radioimmunoassay
• CPC Class Codes: Y10S 436/808 Automated or kit
• CPC Class Codes: Y10S 436/813 Cancer
• CPC Class Codes: Y10S 436/814 Pregnancy
• CPC Class Codes: Y10S 436/815 Test for named compound or class of compounds
• CPC Class Codes: Y10S 436/819 Multifunctional antigen or antibody

Abstract

The invention relates to an immunometric assay for a multivalent antigen in a sample which comprises forming a complex of the antigen together with multiple immobilized monoclonal antibodies against different epitopes of the antigen and with a detectably labeled soluble monoclonal antibody which is identical to one of the multiple immobilized antibodies. The labeled antibody associated with the complex is separated from the remaining soluble antibody and the detectably labeled antibody associated with the complex or unassociated with the complex is detected. Any one of the multiple immobilized monoclonal antibodies shows, by itself, substantially less binding towards the antigen in the immunometric assay, when used with itself or another monocolonal antibody in soluble labeled form, than when used with the multiple immobilized antibodies in combination.

Claims

1. An immunometric assay for a multivalent antigen in a sample, comprising:

(a) forming a mixture of said sample with multiple immobilized monoclonal antibodies against different epitopes on said antigen, and incubating said mixture for a time and under conditions sufficient to allow said antigen in said sample to bind to said multiple immobilized monoclonal antibodies;

(b) adding to said mixture after said incubation of step (a) a detectably labeled monoclonal soluble antibody which is identical to one of said multiple immobilized monoclonal antibodies, and incubating the new resulting mixture for a time and under conditions sufficient to allow said detectably labeled monoclonal soluble antibody to bind to said antigen;

(c) separating detectably labeled monoclonal antibody bound to said antigen from detectably labeled monoclonal soluble antibody; and

(d) detecting either said detectably labeled monoclonal antibody bound to said antigen, or detecting detectably labeled monoclonal soluble antibody not bound to said antigen;

wherein any one of said multiple immobilized monoclonal antibodies shows, by itself, substantially less binding towards said antigen in said immunometric assay, when used with itself or another monoclonal antibody in soluble labeled form, than when used with multiple immobilized monoclonal antibodies in combination.

2. The assay of claim 1, wherein said antigen is alpha-fetoprotein.

3. The assay of claim 1, wherein said antigen is human chorionic gonadotropin.

4. The assay of claim 2, wherein said multiple immobilized monoclonal antibodies comprise at least two immobilized monoclonal antibodies present on the same solid surface, the first of said antibodies having the binding specificity of antibody AF01 obtained from the hybridoma cell line I-295;

and the second of said antibodies having the binding specificity of antibody AF03 obtained from hybridoma cell line I-296.

5. The assay of claim 3, wherein said multiple immobilized monoclonal antibodies comprise at least two immobilized monoclonal antibodies present on the same solid surface, the first of said antibodies having the binding specificity of antibody FB03 obtained from the hybridoma cell line I-297;

and the second of said antibodies having the binding specificity of antibody D1E8 obtained from hybridoma cell line I-298.

6. A kit useful for the detection of a multivalent antigen in a sample comprising a carrier being compartmentalized to receive in close confinement therein one or more containers wherein

(a) a first container contains multiple monoclonal antibodies against different epitopes on said antigen, immobilized on an insoluble solid phase;

(b) a second container contains detectably labelled monoclonal soluble antibody which is identical to one of said multiple immobilized antibodies.

7. The kit of claim 6 wherein said antigen is alphafetoprotein or human chorionic gonadotropin.

8. The kit of claim 6 wherein said first container contains antibodies derived from cell lines I-295 and I-296 and wherein said second container contains antibody derived from cell line I-295.

9. The kit of claim 6 wherein said first container contains antibodies derived from cell lines I-297 and I-298 and wherein said second container contains antibody derived from cell line I-297.

10. An insoluble immunological reagent support for use in an immunometric assay for a multivalent antigen selected from the group consisting of alpha-fetoprotein and human chorionic gonadotropin, which comprises an insoluble support comprising at least two different immobilized monoclonal antibodies against said antigen, wherein said monoclonal antibodies are bound to said insoluble support in a non-interfering manner.

11. The reagent of claim 10 wherein one of said antibodies is derived from hybridoma cell line I-295, which secretes antibody AF01, and the other is derived from hybridoma cell line I-296, which secretes antibody AF03.

12. The reagent of claim 10 wherein one of said antibodies is derived from hybridoma cell line I-297, which secretes antibody FB03, and the other is derived from hybridoma cell line I-298, which secretes antibody D1E8.