Immunoassays in capillaries

• US 5,624,850 A
• Filed: 06/06/1994
• Issued: 04/29/1997
• Est. Priority Date: 06/06/1994
• Status: Expired due to Term

• Current Assignee: IDEXX Laboratories Incorporated
• Original Assignee: Idetek, Inc. (IDEXX Laboratories Incorporated)
• Inventors: Rocco, Richard, Kharadia, Shanta, Deshpande, Sudhir, Piani, Marcos, Kumar, Amit
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): GRASER, JENNIFER E
• Application Number:
  US08/254,302
• Time in Patent Office: 1,058 Days
• Field of Search: 436/518, 436/524, 436/527, 436/536, 436/538, 436/541, 436/20, 436/23, 436/56, 436/164, 436/172, 436/800, 436/805, 436/824, 422/55, 422/57, 422/61, 422/68.1, 422/74, 422/82.07, 422/82.08, 422/312, 435/809, 435/810, 435/968, 435/971, 435/972
• US Class Current: 436/527
• CPC Class Codes: G01N 2021/0346 Capillary cells; Microcells
• CPC Class Codes: G01N 21/03 Cuvette constructions
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/552 Glass or silica
• CPC Class Codes: G01N 33/94 involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
• CPC Class Codes: G01N 35/00 Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor

Abstract

Immunoassay methods employing capillary containers are provided. The immunoassays may be competitive or sandwich immunoassays, where fluorescently labeled conjugates are used. Sample suspected of containing analyte is combined with fluorescently labeled conjugate, as well as any additional reactants which may be required. At least a portion of the incubated sample is placed in a capillary container. The capillary container has, on at least one region of its inner surface, a binding member capable of complexing with the fluorescently labeled conjugate, either directly or indirectly. Fluorescently labeled conjugate not complexed to binding member is then washed from the capillary container. The complexed fluorescently labeled conjugate is detected by irradiating the capillary and measuring the emitted signal. The intensity of the emitted signal is indicative of the presence of analyte in the sample.

Claims

1. An immunoassay method for detecting an analyte in a liquid sample, said method comprising steps of:

providing a first specific binding pair member conjugated to a fluorescent label;

providing a translucent capillary having an inner diameter of from about 0.1 μ

m to 1.0 mm comprising in a detection region a second specific binding pair member, wherein said first specific binding pair member is capable of;

(a) competing with said analyte for binding to said second specific binding pair member or (b) concomitantly binding with said second specific binding pair member to said analyte;

combining said sample with said first member and said second member, whereby said first member binds to said second member in relation to the amount of said analyte in said sample in said detection region, wherein said sample contacts less than 30% of the length of the outer surface of said capillary;

washing away any first member in said capillary not specifically bound to said second member;

irradiating said detection region from a location external to the diameter of the capillary; and

detecting the amount of fluorescence passing through a wall of the capillary from said detection region as indicative of the presence of said analyte in said sample.

2. A method according to claim 1, wherein said combining comprises:

adding said first member to said sample to provide a reaction medium; and

taking up at least a portion of said reaction medium into the capillary, wherein said reaction medium contacts said second member in said detection region.

3. A method according to claim 2, wherein said capillary lacks said second member at a first end and said sample is taken up at said first end comprising the additional step of turning over the capillary, whereby said sample moves to said detection region.

4. A method according to claim 1, wherein said capillary is glass.

5. An immunoassay method for detecting an analyte in a liquid sample, said method comprising steps of:

providing a first specific binding pair member which is competitive with said analyte for binding to the complementary binding member of said pair, said first member conjugated to a fluorescent label;

providing a translucent capillary having an inner diameter of from about 0.1 μ

m to 1.0 mm comprising in a detection region on the internal surface of said capillary a second specific binding pair member capable of binding to said first member;

combining said sample with said first member and said second member, whereby said first member binds to said second member in relation to the amount of said analyte in said sample in said detection region, wherein said sample contacts less than 30% of the length of the outer surface of said capillary;

washing away any first member in said capillary not specifically bound to said second member;

irradiating said detection region from a location external to the diameter of the capillary; and

detecting the amount of fluorescence passing through a wall of the capillary from said detection region as indicative of the presence of said analyte in said sample.

6. A method according to claim 5, wherein said analyte is a hapten and bound in said detection region as a hapten protein conjugate.

7. A method according to claim 6, wherein said first conjugate is an antibody to said hapten.

8. A method according to claim 5, wherein said detection region is distal to a first end of said capillary and wherein said reaction medium is taken up at said first end and said capillary is inverted to move said reaction medium to said detection region.

9. A method according to claim 5, wherein said capillary is glass and the internal surface of said capillary is coated with an aminoalkylsiloxane.

10. An immunoassay method for detecting an haptenic analyte in a liquid sample, said method comprising steps of:

providing an anti-analyte conjugated to a fluorescent label;

providing a translucent capillary having an inner diameter of from about 0.1 μ

m to 1.0 mm coated internally with an aminoalkylsiloxane and comprising a capture hapten conjugated to a protein bound to said capillary internally in a reaction region;

combining said sample with said anti-analyte to, form a reaction medium;

taking up said reaction medium into said capillary where said anti-analyte binds to said capture hapten in relation to the amount of said haptenic analyte in said sample in said detection region, wherein said reaction medium contacts less than 30% of the length of the outer surface of said capillary;

washing away said anti-analyte in said capillary not specifically bound to said capture hapten;

irradiating said detection region from a location external to the diameter of the capillary; and

detecting the amount of fluorescence passing through a wall of the capillary from said detection region, as indicative of the presence of said haptenic analyte in said sample.

11. A method according to claim 10, wherein said haptenic analyte is an antibiotic and said sample is milk.

12. A method according to claim 10, wherein said detection region is distal to a first end of said capillary and wherein said reaction medium is taken up at said first end and said capillary is inverted to move said reaction medium to said detection region.

13. A glass capillary comprising an internal coating of an aminoalkylsiloxane, a protein conjugate coated region extending from a first end only a portion of the length of said capillary and coating an external region extending from said first end not greater than said portion, wherein said protein conjugate is a hapten covalently bonded to a protein.

14. A glass capillary according to claim 13, wherein said hapten is an antibiotic.