Differential separation assay

• US 5,137,609 A
• Filed: 01/31/1992
• Issued: 08/11/1992
• Est. Priority Date: 01/31/1992
• Status: Expired due to Fees

• Current Assignee: Biometric Imaging, Inc. (Becton, Dickinson & Co)
• Original Assignee: Biometric Imaging, Inc. (Becton, Dickinson & Co)
• Inventors: Manian, Bala S., Ghazarossian, Vartan
• Primary Examiner(s): Niebling, John
• Assistant Examiner(s): STARSIAK, JOHN S
• Application Number:
  US07/828,407
• Time in Patent Office: 193 Days
• Field of Search: 204/299 R, 204/182.8, 204/182.7, 204/182.9, 204/180.1, 356/344
• US Class Current: 204/452
• CPC Class Codes: G01N 27/44721 by optical means

Abstract

An electrophoresis-based assay system for detection of one or more target substances, i.e. an analyte tagged with fluorescent binding agents. The analyte is reacted with an excess amount of fluroescently tagged binding agent. The reaction mixture is subjected to electrophoresis and the migration of bound and free fluorescent substances are timed at a location where there is a spatial and optical differentiation of the two substances. An optical detector supplies signals corresponding to fluorescent amplitudes of the two substances. The free fluorescent substance arrives at a time expected from calibration runs. This optical signal is a marker for a second time, either earlier or later, when the bound substance should have arrived. Recorded data is searched to establish the relation between free and bound dye among the recorded optical signals. An absence of a bound dye signal infers the absence of target analyte in a sample. The amounts of bound and unbound amounts of the same fluorescent substance may be related at ratio of amplitudes of the optical signals so that the amount of target analyte may be estimated.

Claims

1. A method for detecting target substances during electrophoresis comprising,

mixing a specific binding agent with a sample which may contain a target substance, the binding agent being specific to the target substance and having a fluorescent characteristic, the amount of binding agent in excess of what will react with the target substance so that there is free and bound binding agent in the sample, both the bound and free binding agent having expected electrophoretic migration times from a starting location to a measuring place,

conducting electrophoresis of said mixture in a path defined by electrodes,

measuring and recording the times wherein substances having said fluorescent characteristic reach the measuring place,

searching said recorded times for bound binding agent in relation to free binding agent using said expected electrophoretic migration times in comparison to said measured times wherein finding of said bound binding agent indicates presence of said target substance.

2. The method of claim 1 wherein said binding agent is selected from the group consisting of a fluorescent antibody, antigen, receptor, enzyme substrate, enzyme inhibitor and lectin.

3. The method of claim 1 wherein said binding agent is conjugated with a fluorescent dye.

4. The method of claim 1 wherein said measuring place is a slit along an electrophoretic trajectory of said bound and unbound binding agent.

5. A method for detecting target substances during electrophoresis comprising,

(a) reacting one or more known target analytes each with an excess amount of a characteristic fluorescent substance, creating a mixture of bound and free fluorescent substance,

(b) causing electrophoretic migration in a gel of the bound and free fluorescent substance starting from a known starting position, the fluorescent substance migrating toward an electrode along a path,

(c) directing actinic radiation to said fluorescent substance, thereby causing fluorescence,

(d) detecting the characteristic fluorescence of the unbound fluorescent substance at a fixed position on said path,

(e) detecting the characteristic fluorescence of the fluorescent substance bound to the analyte at a time different from the time of the free substance measurement at said fixed position, thereby creating a time difference,

(f) recording the time difference to form a time window linking the travel times of the bound and free substances past said fixed position,

(g) repeating steps (a)-(e) for patient samples in which the presence of target analyte is not known,

(h) using the time window to associate bound fluorescent substance with free fluorescent substance in order to establish the presence of target analyte in the patient samples.

6. The method of claim 5 wherein said reacting of target analyte is by means of a fluorescent dye or dye embedded in beads or microspheres attached to binding agent.

7. The method of claim 5 wherein said binding agent is selected from the group consisting of a fluorescent antibody, antigen, receptor, enzyme substrate, enzyme inhibitor and lectin.

8. The method of claim 5 wherein said electrophoretic migration is within a single lane.

9. The method of claim 5 wherein said electrophoretic migration is within a plurality of lanes, one or more of which is used for a known amount of target analyte.

10. The method of claim 9 where the lanes are gel filled capillaries or cuvettes.

11. The method of claim 5 wherein the area under each peak of each detected characteristic fluorescence is measured.

12. The method of claim 6 wherein said detection of the amplitude of characteristic fluorescence is measured through a slit or pinhole as analytes migrate past said slit or pinhole.

13. The method of claim 5 wherein said time window is formed with approximately one statistical variance.