US4421860A
Expired / Lapsed
US
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Homogeneous fluoroimmunoassay involving autocorrelation processing of optically sensed signals
Assignee: University of California
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Filing: 1981-10-13
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Granted: 1983-12-20
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Expires: 2001-10-13
Abstract
Abstract In the course of a reaction in which one of the reactants is on the surface of carrier particles in a solution and another of the reactants is tagged with a fluorescent substance, some of the fluorescently tagged reactant attaches to, or is displaced from the carrier particle. The present invention relates to a method and device for determining the amount of fluorescently-tagged reactant which is attached to the carrier particle or which is free in solution, without physically separating the carrier particles from the solution. In a particular application of the invention (immunoassay) the reaction is between antibodies and antigens, and from the amount of fluorescently-tagged reactant which is attached to the carrier particle one can determine the unknown amount of antigen in a sample. The number of fluorescent particles which are bound to the large carrier particles and which are in solution are determined by optical and electronic sensing and analyzing techniques, including (1) shadowing techniques in which large opaque carrier particles block the pick-up of fluorescent radiation from bound tagged reactants, and/or (2) autocorrelation techniques which selectively discriminate between radiation from the bound fluorescently tagged reactants on the large particles and the free fluorescently-tagged reactants which have a more rapid diffusion movement than the carrier particles.
Inventors
- 👤 Virgil B. Elings
- 👤 David F. Nicoli
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-26 00:32
CPC Classifications
G; G01; G01N; G01N21/00; G01N21/62; G01N21/63; G01N21/64; G01N21/6428; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/536; G01N33/542; G01N15/00; G01N15/02; G01N15/0205; G01N15/0211; G01N2015/0216; G01N2015/0222; Y; Y10; Y10S; Y10S436/00; Y10S436/80; Y10S436/807
Claims Analysis
Total Claims: 22
Claim 1
Independent Claim
198 words
A homogeneous fluorescent immunoassay method comprising the steps of: providing relatively large carrier particles in solution with antibodies residing at a plurality of sites on each of said carrier particles; providing at least first and second active components; with the first being an unknown antigen to be tested, the second being tagged with a fluorescent substance; exposing said carrier particles to the first and second components, to cause a reaction between said first and second components and the antibodies on said particles; whereby the number of fluorescently tagged components bonded to said carrier particles changes; illuminating the solution containing both the free and bound fluorescently tagged substances with optical radiation at a first wavelength to cause fluorescent output radiation at a second longer wavelength; optically sensing output signals at said second wavelength resulting from said fluorescent output radiation; and electronically processing the optically sensed signals to determine the amount of the fluorescently tagged component which is free and/or that which is bound to the carrier particles without physically separating the free and bound fluorescent material, said electronic processing including autocorrelation processing to discriminate between fluorescent radiation arising from the bound fluorescent material and from the free fluorescent material.
Claim 2
Dependent Claim
198 words
A homogeneous fluorescent immunoassay method comprising the steps of: providing relatively large carrier particles in solution with antibodies residing at a plurality of sites on each of said carrier particles; providing at least first and second active components; with the first being an unknown antigen to be tested, the second being tagged with a fluorescent substance; exposing said carrier particles to the first and second components, to cause a reaction between said first and second components and the antibodies on said particles; whereby the number of fluorescently tagged components bonded to said carrier particles changes; illuminating the solution containing both the free and bound fluorescently tagged substances with optical radiation at a first wavelength to cause fluorescent output radiation at a second longer wavelength; optically sensing output signals at said second wavelength resulting from said fluorescent output radiation; and electronically processing the optically sensed signals to determine the amount of the fluorescently tagged component which is free and/or that which is bound to the carrier particles without physically separating the free and bound fluorescent material, said electronic processing including autocorrelation processing to discriminate between fluorescent radiation arising from the bound fluorescent material and from the free fluorescent material.
Claim 3
Dependent Claim
19 words
A fluorescent immunoassay method as defined in claim 1 wherein the autocorrelation function at zero sampling time is used.
Claim 4
Dependent Claim
46 words
A fluorescent immunoassay method as defined in claim 1 including the step of selecting a very small volume for sampling to include a relatively small number of the larger carrier particles, so that the relative fluctuation in the number of such larger particles will be large.
Claim 5
Dependent Claim
30 words
A fluorescent immunoassay method as defined in claim 1 wherein the electronic processing includes measuring the fluctuations in the fluorescent radiation to emphasize the fluorescence from the brighter carrier particles.
Claim 6
Dependent Claim
48 words
A fluorescent immunoassay method as defined in claim 1 in which the second component is an antigen of the same kind as the first component and the carrier particle is exposed to both the first and second components which then compete for binding sites on the carrier particle.
Claim 7
Dependent Claim
34 words
A fluorescent immunoassay method as defined in claim 1 in which the second component is an antibody and the carrier particle is initially exposed to the first component and then to the second component.
Claim 8
Dependent Claim
20 words
A fluorescent immunoassay method as defined in claim 1 wherein said carrier particles are biological entities having natural reactive sites.
Claim 9
Dependent Claim
45 words
A fluorescent immunoassay method as defined in claim 1 including the steps of using opaque carrier particles, and sensing fluorescent radiation from the "forward" direction relative to said first wavelength radiation, whereby the carrier particles shield or "shadow" the radiation from the bound fluorescent molecules.
Claim 10
Dependent Claim
59 words
A fluorescent immunoassay method as defined in claim 8 wherein the fluorescent radiation is also sensed in the "back" direction, to obtain full fluorescent radiation from the bound fluorescent molecules, and further including the steps of comparing the signals sensed in the "forward" and in the back directions to determine the relative levels of bound and free fluorescent molecules.
Claim 11
Dependent Claim
42 words
A fluorescent immunoassay method as defined in claim 1 including the steps of selecting a series of very small volumes to include a relatively small number of said large carrier particles in each volume, and sampling the fluorescence from each said volume.
Claim 12
Dependent Claim
32 words
A fluorescent immunoassay method as defined in claim 10 wherein each of said volumes is only sampled once prior to determining the amount of the free and the bound fluorescently tagged component.
Claim 13
Dependent Claim
34 words
A fluorescent immunoassay method as defined in claim 10 wherein each of said volumes is sampled a plurality of times prior to determining the amount of the free and the bound fluorescently tagged component.
Claim 14
Dependent Claim
242 words
A homogeneous fluorescent immunoassay method comprising the steps of: providing carrier particles of a relatively large size in solution and with a large number of reactive sites on the surface of each particle; providing at least first and second active components; with one being of known properties and composition and the other being an unknown to be tested; with one being tagged with a fluorescent substance and the other being untagged; and said first and second components including antigens and antibodies; exposing said carrier particles to said first and second component, to cause a reaction between said first and second components and the reactive sites on the carrier particles; whereby the number of fluorescently tagged antibodies or antigens bonded to said carrier particles changes; illuminating the solution containing both the free and bound fluorescently tagged substances with optical radiation at a first frequency to cause fluorescent output radiation at a second different frequency; optically sensing output signals at said second frequency, resulting from said fluorescent output; electronically processing the optically sensed signals to determine the amount of the fluorescently tagged component which is free and which is bound to the carrier particles without physically separating the free and bound fluorescent material; and selecting a very small volume for sampling to include a relatively small number of the larger carrier particles so that the relative fluctuation in the number of such large particles in the volume causes a big fluctuation in output fluorescence.
Claim 15
Dependent Claim
242 words
A homogeneous fluorescent immunoassay method comprising the steps of: providing carrier particles of a relatively large size in solution and with a large number of reactive sites on the surface of each particle; providing at least first and second active components; with one being of known properties and composition and the other being an unknown to be tested; with one being tagged with a fluorescent substance and the other being untagged; and said first and second components including antigens and antibodies; exposing said carrier particles to said first and second component, to cause a reaction between said first and second components and the reactive sites on the carrier particles; whereby the number of fluorescently tagged antibodies or antigens bonded to said carrier particles changes; illuminating the solution containing both the free and bound fluorescently tagged substances with optical radiation at a first frequency to cause fluorescent output radiation at a second different frequency; optically sensing output signals at said second frequency, resulting from said fluorescent output; electronically processing the optically sensed signals to determine the amount of the fluorescently tagged component which is free and which is bound to the carrier particles without physically separating the free and bound fluorescent material; and selecting a very small volume for sampling to include a relatively small number of the larger carrier particles so that the relative fluctuation in the number of such large particles in the volume causes a big fluctuation in output fluorescence.
Claim 16
Dependent Claim
35 words
A fluorescent immunoassay method as defined in claim 13 wherein said electronic processing includes autocorrelation processing to disciminate between fluorescent radiation arising on the larger particles and the fluorescent radiation from smaller "free" fluorescent molecules.
Claim 17
Dependent Claim
35 words
A fluorescent immunoassay method as defined in claim 13 including the step of filtering the fluorescent radiation at said second frequency prior to optical sensing to eliminate scattered or direct radiation at said first frequency.
Claim 18
Dependent Claim
58 words
A fluorescent immunoassay method as defined in claim 13 including the step of selecting a series of very small volumes for sampling to include a relatively small number of said larger carrier particles in each sample, so that variations in the number of said larger particles in said volumes will cause large fluctuations in the sensed output fluorescence.
Claim 19
Dependent Claim
30 words
A fluorescent immunoassay method as defined in claim 13 wherein the electronic processing includes measuring the fluctuations in the fluorescent radiation to emphasize the fluorescence from the brighter carrier particles.
Claim 20
Dependent Claim
19 words
A fluorescent immunoassay method as defined in claim 17 where the autocorrelation function at zero sampling time is used.
Claim 21
Dependent Claim
45 words
A fluorescent immunoassay method as defined in claim 13 including the steps of using opaque carrier particles, and sensing fluorescent radiation from the "forward" direction relative to said first frequency radiation, whereby the carrier particles shield or shadow the radiation from the bound fluorescent molecules.
Claim 22
Dependent Claim
59 words
A fluorescent immunoassay method as defined in claim 19 wherein the fluorescent radiation is also sensed in the "back" direction, to obtain full fluorescent radiation from the bound fluorescent molecules, and further including the steps of comparing the signals sensed in the "forward" and in the back directions to determine the relative levels of bound and free fluorescent molecules.
References Cited (Backward Citations)
US4275149A
Cited
US4318707A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1986-12-19 | 1987-06-20 | 1987-12-20 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1990-12-19 | 1991-06-20 | 1991-12-20 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1994-12-19 | 1995-06-20 | 1995-12-20 | $7700.00 | Window Closed / Lapsed |