Double capture assay method employing a capillary flow device

• US 5,145,784 A
• Filed: 01/02/1992
• Issued: 09/08/1992
• Est. Priority Date: 05/04/1988
• Status: Expired due to Fees

• Current Assignee: Cambridge BioScience Corp. (Agenus Inc.)
• Original Assignee: Cambridge Biotech Corp. (Agenus Inc.)
• Inventors: Neri, Bruce P., Cox, Daniel E., Shinefeld, Lisa, Bauminger, Sara, Abril, Obsidiana
• Primary Examiner(s): Johnston, Jill A.
• Application Number:
  US07/816,772
• Time in Patent Office: 250 Days
• Field of Search: 436/523, 436/526, 436/816, 436/901
• US Class Current: 436/526
• CPC Class Codes: G01N 33/54333 Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
• CPC Class Codes: G01N 33/585 with a particulate label, e.g. coloured latex
• CPC Class Codes: G01N 33/946 CNS-stimulants, e.g. cocaine, amphetamines
• CPC Class Codes: G01N 35/0098 involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation magnetic particles used in immunoassays G01N33/54326; magnetic separation in general B03C
• CPC Class Codes: Y10S 436/816 Alkaloids, amphetamines, and barbiturates
• CPC Class Codes: Y10S 436/901 Drugs of abuse, e.g. narcotics, amphetamine

Abstract

A capillary flow device useful in double capture assays, such as double capture immunoassays, and a double capture assay. The capillary flow device comprises a capillary track, a sample receptacle, a particle collecting means and, optionally, a magnet and a particle concentrator. The assay method is useful for determining any analyte of interest for which there is a specific binding partner.

Claims

1. A double capture immunoassay for an antigen of interest in a sample, comprising the steps of:

a. combining;

     1) the sample;

     2) magnetic particles having affixed thereto antigen of interest;

     3) nonmagnetic detectable particles having affixed thereto the antigen of interest; and

     4) unbound antibody specific for the antigen of interest, to produce a reaction mixture;

b. incubating the reaction mixture under conditions and for a period of time sufficient for antigen of interest to bind to antibody specific for the antigen;

c. separating the magnetic particles from the nonmagnetic particles;

d. capturing the nonmagnetic particles on a solid material; and

e. determining the presence or absence of captured nonmagnetic particles on the solid material.

f. determining the presence or absence of the antigen of interest in the sample by relating the presence or absence of captured nonmagnetic particles to a relationship between captured nonmagnetic detectable particles having affixed thereto the antigen of interest and the antigen of interest.

2. A double capture immunoassay of claim 1 wherein the antigen of interest is quantitated by determining the number of captured nonmagnetic particles on the solid material and relating the number of captured nonmagnetic particles present on the solid material to a predetermined quantitative relationship between the number of captured nonmagnetic particles and the quantity of antigen of interest.

3. A double capture immunoassay for an antibody of interest in a sample, comprising the steps of:

a. combining;

     1) the sample;

     2) magnetic particles having affixed thereto antigen for which the antibody of interest is specific; and

     3) nonmagnetic detectable particles having affixed thereto antigen for which the antibody of interest is specific, to produce a reaction mixture;

b. incubating the reaction mixture under conditions and for a period of time sufficient for antibody of interest to bind to antigen for which it is specific;

c. separating the magnetic particles from the nonmagnetic particles;

d. capturing the nonmagnetic particles on a solid material;

e. determining the presence or absence of captured nonmagnetic particles on the solid material.

f. determining the presence or absence of the antibody of interest in the sample by relating the presence or absence of captured nonmagnetic particles to a relationship between captured nonmagnetic detectable particles having affixed thereto the antigen for which the antibody of interest is specific and the antibody of interest.

4. A double capture immunoassay of claim 3 wherein the antibody of interest is quantitated by determining the number of captured nonmagnetic particles on the solid material and relating the number of captured nonmagnetic particles present on the solid material to a predetermined quantitative relationship between the number of captured nonmagnetic particles and the quantity of antibody of interest.

5. A double capture assay for a drug of abuse in a sample, comprising the steps of:

a. combining;

     1) the sample;

     2) magnetic particles having affixed thereto the drug of abuse or a metabolite thereof;

     3) nonmagnetic detectable particles having affixed thereto the drug of abuse or a metabolite thereof; and

     4) specific binding partner for the drug of abuse or a metabolite thereof, to produce a reaction mixture;

b. incubating the reaction mixture under conditions and for a period of time sufficient for the drug of abuse and the specific binding partner to bind;

c. separating the magnetic particles from the nonmagnetic particles;

d. capturing the nonmagnetic particles; and

e. determining the presence or absence of captured particles.

f. determining the presence or absence of the drug of abuse in the sample by relating the presence or absence of captured nonmagnetic particles to a relationship between captured nonmagnetic detectable particles having affixed thereto the drug of abuse or a metabolite thereof and the drug of abuse.

6. A double capture assay of claim 5 wherein the drug of abuse is cocaine, the metabolite thereof is benzoylecgonine, and the specific binding partner is monoclonal anti-benzoylecgonine.

7. An assay for detecting an analyte of interest which is a member of a specific binding pair in a sample, comprising the steps of:

a) combining the following components;

i) the sample;

ii) magnetic particles having affixed thereto a first member of a specific binding pair;

iii) non-magnetic detectable particles having affixed thereto a second member of a specific binding pair; and

b) incubating the mixture from step a) under conditions appropriate for the binding of complementary members of a specific binding pairs;

c) separating the magnetic particles, and non-magnetic particles bound thereto, from unbound non-magnetic particles;

d) capturing the unbound non-magnetic particles; and

e) determining the presence of captured unbound non-magnetic particles.

f. determining the presence or absence of the analyte of interest in the sample by relating the presence or absence of captured nonmagnetic particles to a relationship between captured nonmagnetic detectable particles having affixed thereto the second member of the specific binding pair and the analyte of interest.

8. A method of claim 7 wherein steps b), c) and d) are carried out in a capillary flow device comprising:

a) a capillary track having a first and a second distal end;

b) a sample receptacle in communication with the first distal end of the capillary track; and

c) a liquid reservoir at the second distal end of the capillary track.

9. A method of claim 8 wherein the capillary flow device further comprises:

d) a particle concentrator in communication with the second distal end of the capillary track.

10. An assay of claim 7 wherein the amount of the analyte of interest present in the sample is quantitated by determining the number of captured unbound non-magnetic particles and relating the number of captured unbound non-magnetic particles to a predetermined quantitative relationship between the number of captured non-magnetic particles and the quantity of the analyte of interest.

11. An assay of claim 7 wherein the analyte of interest is an antigen specifically reactive with an antibody, the first member of a specific binding pair is an antigen specifically reactive with the antibody and the second member of a specific binding pair is the antibody.

12. An assay of claim 7 wherein the analyte of interest is an antigen specifically reactive with an antibody, the first member of a specific binding pair is the antibody and the second member of a specific binding pair is an antigen specifically reactive with the antibody.

13. An assay of claim 7 wherein the analyte of interest is an antigen, the first member of a specific binding pair is an antibody specifically reactive with the antigen and the second member of a specific binding pair is an antibody specifically reactive with the antigen.

14. An assay of claim 7 wherein the analyte of interest is an antibody and the first member of a specific binding pair is an antigen specifically reactive with the antibody and the second member of a specific binding pair is an antigen specifically reactive with the antibody.

15. An assay of claim 7 wherein the analyte of interest is an antibody and the first member of a specific binding pair is an antigen specifically reactive with the antibody and the second member of a specific binding pair is an antibody specifically reactive with the antigen.

16. An assay of claim 7 wherein the analyte of interest is an antibody specifically reactive with an antigen and the first member of a specific binding pair is an antibody specifically reactive with the antigen and the second member of a specific binding pair is the antigen.

17. An assay for detecting an analyte of interest which is a member of a specific binding pair in a sample, comprising the steps of:

a) combining the following components;

i) the sample;

ii) magnetic particles having affixed thereto a first member of a specific binding pair;

iii) non-magnetic detectable particles having affixed thereto a second member of a specific binding pair; and

iv) a third member of a specific binding pair specifically reactive with the analyte of interest; and

b) incubating the mixture from step a) under conditions appropriate for binding of complementary members of specific binding pairs;

c) separating the magnetic particles, and non-magnetic particles bound thereto, from unbound non-magnetic particles;

d) capturing the unbound non-magnetic particles; and

e) determining the presence of captured unbound non-magnetic particles.

f. determining the presence or absence of the analyte of interest in the sample by relating the presence or absence of captured non-magnetic particles to a relationship between captured non-magnetic detectable particles having affixed thereto the second member of the specific binding pair and the analyte of interest.

18. A method of claim 17 wherein steps b), c) and d) are carried out in a capillary flow device comprising:

a) a capillary track having a first and a second distal end;

b) a sample receptacle in communication with the first distal end of the capillary track; and

c) a liquid reservoir at the second distal end of the capillary track.

19. A method of claim 18 wherein the capillary flow device further comprises:

d) a particle concentrator in communication with the second distal end of the capillary track.

20. An assay of claim 17 wherein the amount of the analyte of interest present in the sample is quantitated by determining the number of captured unbound non-magnetic particles and relating the number of captured unbound non-magnetic particles to a predetermined quantitative relationship between the number of captured non-magnetic particles and the quantity of the analyte of interest.

21. An assay of claim 17 wherein the analyte of interest is an antigen reactive with an antibody, the first member of a specific binding pair is an antigen specifically reactive with the antibody, the second member of a specific binding pair is antigen specifically reactive with the antibody and the third member of a specific binding pair is the antibody.

22. An assay of claim 17 wherein the analyte of interest is an antibody specifically reactive with an antigen, the first member of a specific binding pair is an antibody specifically reactive with the antigen, the second member of a specific binding pair is an antibody specifically reactive with the antigen, and the third member of a specific binding pair is the antigen.

23. An assay of claim 17 wherein the analyte of interest is an antigen, the first member of a specific binding pair is an antibody specifically reactive with the antigen, the second member of a specific binding pair is an antibody specifically reactive with the antigen and the third member of a specific binding pair is an antigen specifically reactive with the first member of a specific binding pair and the second member of a specific binding pair.