Immunoassays and kits for use therein which include low affinity antibodies for reducing the hook effect

• US 4,595,661 A
• Filed: 11/18/1983
• Issued: 06/17/1986
• Est. Priority Date: 11/18/1983
• Status: Expired due to Term

• Current Assignee: Beckman Instruments, Inc. (Danaher Corporation)
• Original Assignee: Beckman Instruments, Inc. (Danaher Corporation)
• Inventors: Harris, Paul C., Lee, Shih-Yun, Tung, Ker-Kong, Vodian, Morton A., Cragle, Linda K.
• Primary Examiner(s): Marantz, Sidney
• Assistant Examiner(s): WHITE, PATRICIA K
• Application Number:
  US06/553,219
• Time in Patent Office: 942 Days
• Field of Search: 436/513, 436/518, 436/531, 436/534, 436/536, 436/538, 436/548, 436/805, 436/808, 436/818, 436/819, 436/533, 435/4, 435/7, 435/810
• US Class Current: 435/7.94
• 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 33/54393 Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• 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: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/962 Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/808 Automated or kit
• CPC Class Codes: Y10S 436/818 Human chorionic gonadotropin
• CPC Class Codes: Y10S 436/819 Multifunctional antigen or antibody

Abstract

An immunoassay for assaying an antigenic substance (Ag) in a fluid. The immunoassay is the type which comprises contacting the fluid with at least one first entity selected from a group consisting of an antibody (Ab) to the Ag, a soluble, labeled antibody (L-Ab_a) to the Ag, and an antibody (Ab_b) to the Ag bound to a solid support (SC). The immunoassay is characterized in that the fluid is contacted with at least one additional entity selected from a group consisting of at least one different type of soluble, labeled antibody (L-Ab_c) to the Ag, at least one different type of antibody (Ab_d) bound to a solid carrier (SC₁), and at least one different type of antibody (Ab_e) to the Ag. The SC₁ is selected from a group consisting of SC, at least one different solid carrier (SC₂), and mixtures thereof (SC and SC₂). Each type of L-Ab_c, Ab_d -SC₁, and Ab_e has a lower average affinity constant (K) for Ag than each respective K of L-Ab_a, Ab_b -SC and Ab.sub. e ; and the additional entity is present in an amount sufficient to avoid a hook effect.

Also, a reagent of the type comprising at least one first entity selected from a group consisting of L-Ab_a, Ab_b -SC, and Ab. The reagent is characterized in that it further comprises at least one additional entity selected from a group consisting of at least one different type of L-Ab_c and at least one different type of Ab_d -SC₁, and at least one different type of Ab_e. Ab, L-Ab_a, Ab_b -SC, L-Ab_c, Ab_d -SC₁, and Ab_e are as defined above.

Claims

1. A one-step sandwich immunoassay for an antigenic substance (Ag) in a fluid, said immunoassay being of the type comprising:

(a) contacting said fluid with (i) a soluble, labeled antibody (L-Ab_a) to said Ag and (ii) an antibody (Ab_b) to said Ag bound to a solid carrier (SC) to form an insoluble complex (L-Ab_a -Ag-Ab_b -SC);

(b) separating said L-Ab_a -Ag-Ab_b -SC from said fluid and unreacted L-Ab_a ; and

(c) measuring either the amount of L-Ab_a associated with said L-Ab_a -Ag-Ab_b -SC or the amount of unreacted L-Ab_a ;

characterized in that in step (a) said fluid is also contacted with at least one additional entity selected from the group consisting of at least one different type of soluble, labeled antibody (L-Ab_c) to said Ag and at least one different type of antibody (Ab_d) to said Ag bound to a solid carrier (SC₁);

wherein;

(i) each type of L-Ab_c and Ab_d -SC₁ has a lower average affinity constant (K) for said Ag than each respective K of L-Ab_a and Ab_b -SC;

(ii) said additional entity is present in an amount sufficient to avoid a hook effect; and

(iii) said SC₁ is selected from the group consisting of said SC, at least one different solid carrier (SC₂), and mixtures thereof (SC and SC₂).

2. The immunoassay of claim 1 wherein said additional entity is said L-Ab_c.

3. The immunoassay of claim 1 wherein Ab_a, Ab_b, Ab_c, and Ab_d are each independently selected from the group consisting of monoclonal antibodies, polyclonal antibodies, and mixtures thereof.

4. The immunoassay of claim 1 wherein Ab_a and Ab_c are monoclonal antibodies.

5. The immunoassay of claim 4 wherein said additional entity is said L-Ab_c.

6. A two-step sandwich immunoassay for an antigenic substance (Ag) in a fluid, said immunoassay being of the type comprising:

(a) contacting said fluid with an antibody (Ab_b) bound to a solid carrier (SC) to form an insoluble complex (SC-Ab_b -Ag);

(b) contacting said SC-Ab_b -Ag with a soluble, labeled antibody (Ab_a -L) in order to form a complex (SC-Ab_b -Ag-Ab_a -L);

(c) separating said SC-Ab_b -Ag-Ab_a -L from unreacted L-Ab_a ; and

(d) measuring either the amount of L-Ab_a associated with said SC-Ab_b -Ag-Ab_a -L or the amount of unreacted L-Ab_a ;

characterized in that in step (b) said SC-Ab_b -Ag is also contacted with at least one different type of soluble, labeled antibody (L-Ab_c);

wherein;

(i) each type of L-Ab_c has a lower average affinity constant (K) for said Ag than the K of L-Ab_a ; and

(ii) said additional entity is present in an amount sufficient to avoid a hook effect.

7. The immunoassay of claim 6 wherein Ab_a, Ab_b, and Ab_c are each independently selected from the group consisting of monoclonal antibodies, polyclonal antibodies, and mixtures thereof.

8. The immunoassay of claim 6 wherein Ab_a and Ab_c are monoclonal antibodies.

9. A direct nephelometric immunoassay for an antigenic substance (Ag) in a fluid, said immunoassay being of the type comprising:

(a) contacting said fluid with an antibody (Ab) to said Ag in order to form a complex Ab-Ag; and

(b) measuring the amount of formed Ab-Ag;

characterized in that in step (a) said fluid is also contacted with at least one additional type of antibody Ab_e ;

wherein;

(i) each type of Ab_e has an average affinity constant (K) for said Ag lower than the K of said Ab; and

(ii) said additional entity is present in an amount sufficient to avoid a hook effect.

10. The direct nephelometric immunoassay of claim 9 wherein each additional type of Ab_e is a monoclonal antibody.

11. The direct nephelometric immunoassay of claim 9 wherein each additional type of Ab_e is a polyclonal antibody.

12. A reagent of the type comprising:

(a) a soluble, labeled antibody (L-Ab_a) to an antigenic substance (Ag); and

(b) an antibody (Ab_b) to said Ag bound to a solid carrier (SC);

characterized in that said reagent further comprises at least one additional entity selected from the group consisting of at least one different type of soluble, labeled antibody (L-Ab_c) to said Ag and at least one different type of antibody (Ab_d) to said Ag bound to a solid carrier (SC₁);

wherein;

(i) each type of L-Ab_c and Ab_d -SC₁ has a lower average affinity constant (K) for said Ag than each respective K of L-Ab_a and Ab_b -SC;

(ii) said additional entity is present in an amount sufficient to avoid a hook effect when said reagent is employed in an immunoassay; and

(iii) said SC₁ is selected from the group consisting of said SC, at least one different solid carrier (SC₂), and mixtures thereof (SC and SC₂).

13. The reagent of claim 12 wherein said Ab_a, Ab_b, Ab_c, and Ab_d are each independently selected from the group consisting of monoclonal antibodies, polyclonal antibodies, and mixtures thereof.

14. The reagent of claim 12 wherein said Ab_a and Ab_c are monoclonal antibodies.

15. The reagent of claim 14 wherein said additional entity is said L-Ab_c.

16. The reagent of claim 12 wherein said additional entity is said L-Ab_c.

17. A reagent of the type comprising an antibody (Ab) to an antigenic substance (Ag), characterized in that said reagent further comprises at least one different type of antibody (Ab_e), wherein:

(i) each different type of said Ab_e has a lower average affinity constant (K) for said Ag than the K of said Ab; and

(ii) said additional entity is present in an amount sufficient to avoid a hook effect when said reagent is employed in an immunoassay.

18. The reagent of claim 17 wherein each of said Ab_e is independently selected from the group consisting of monoclonal antibodies, polyclonal antibodies, and mixtures thereof.

19. The reagent of claim 18 wherein each of said Ab_e is a monoclonal antibody.

20. The reagent of claim 18 wherein each of said Ab_e is a polyclonal antibody.