Automated radioimmunoassay

• US 4,022,577 A
• Filed: 03/12/1976
• Issued: 05/10/1977
• Est. Priority Date: 03/12/1976
• Status: Expired due to Term

• Current Assignee: University of Virginia
• Original Assignee: University of Virginia
• Inventors: Brooker, Gary, Price, Michael G., Terasaki, Wesley L.
• Primary Examiner(s): Serwin, R.E.
• Application Number:
  US05/666,302
• Time in Patent Office: 424 Days
• Field of Search: 23/230 B, 23/253 R, 23/259, 424/1, 424/1.5, 195/127
• US Class Current: 436/517
• CPC Class Codes: G01N 33/5302 Apparatus specially adapted for immunological test procedures
• CPC Class Codes: Y10S 436/804 Radioisotope, e.g. radioimmunoassay
• CPC Class Codes: Y10S 436/808 Automated or kit

Abstract

An automated radioimmunoassay apparatus which comprises:

Means for time controlled incubation of a multiplicity of sample solutions, each of which comprise a mixture of (a) a sample, possibly containing an antigen, intended to be assayed, (b) a solution of a known concentration of an antigen tagged with a radioactive isotope and (c) a solution of a known titer of an antibody reactable with said antigen;

Radioactivity detector means for providing a kinetic measurement of radioactivity as a solution is continuously passed therethrough, and for providing a static measurement of radioactivity as a solution is maintained statically within said detector;

Means for introducing said incubated sample into said radioactivity detector means;

Timing means, which is activatable by said radioactivity detector means when said detector means detects a predetermined threshold of radioactivity, for measuring time sequences and for generating control signals at predetermined time sequences;

Recording means coupled to said detector means for recording the amounts of radioactivity detected in said detector means;

Means for separating said kinetically measured solution into a first portion containing antigen which is unreacted with said antibody, and a second portion containing antigen reacted with said antibody;

Means for introducing one of said portions from said separating means into said radioactivity measuring means, for static detection;

Means for isolating said incubated sample from additional sample solutions introduced into said apparatus for assay, wherein said isolating means is activatable by a control signal emitted from said timing means, whereby said isolating means isolates the system at a first predetermined signal from said timing means, and whereby said isolating means reconnects the system to said incubation means at a second predetermined signal from said timing means, wherein the second predetermined time period is set so as to enable sufficient time to complete said static measurement before the next incubated sample arrives in the said detector means for the next sequential kinetic measurement; and

Means for flushing said sample from said system after static measurement thereof so as to prepare said detector for the next sequential sample for static measurement.

Claims

1. An automated radioimmunoassay apparatus which comprises:

means for time controlled incubation of a multiplicity of sample solutions, each of which comprise a mixture of (a) a sample, possibly containing an antigen, intended to be assayed, (b) a solution of a known concentration of an antigen tagged with a radioactive isotope, and (c) a solution of a known titer of an antisera containing antibodies reactable with said antigen;

radioactivity detector means for providing a kinetic measurement of radioactivity as a solution is continuously passed therethrough, and for providing a static measurement of radioactivity as a solution is maintained statically within said detector;

means for introducing said incubated sample into said radioactivity detector means;

timing means, which is activatable by said radioactivity detector means when said detector means detects a predetermined threshold of ratioactivity for measuring time sequences and for generating control signals at predetermined time sequences;

recording means coupled to said detector means for recording the amounts of radioactivity detected in said detector means;

means for separating said kinetically measured solution into a first portion containing antigen which is unreacted with said antibody, and a second portion containing antigen reacted with said antibody;

means for introducing one of said portions from said separating means into said radioactivity measuring means, for static detection;

means for isolating said incubated sample from additional sample solutions introduced into said apparatus for assay, wherein said isolating means is activatable by a control signal emitted from said timing means, whereby said isolating means isolates the system at a first predetermined signal from said timing means, and whereby said isolating means reconnects the system to said incubation means at a second predetermined signal from said timing means, wherein the second predetermined time period is set so as to enable sufficient time to complete said static measurements before the next incubated sample arrives in the said detection means for the next sequential kinetic measurement of radioactivity.

2. The apparatus of claim 1, wherein said recorder means records the amount of radioactivity in the sample as it is passed kinetically through said detector, and wherein the assay results are obtainable from said recorder as a ratio of said static measurement to said kinetic measurement.

3. The apparatus of claim 1, wherein said detector means comprises a first section which is capable of detecting radioactivity amounts as a sample is kinetically passed therethrough and a second section which is capable of detecting radioactivity amounts as a sample solution is held in a substantially static position.

4. The apparatus of claim 1, wherein means are provided for increasing the rate of flow of the said incubated sample through said separating means, said means being activated by a signal from said timing means after said kinetic measurement.

5. The apparatus of claim 1, which further comprises:

means for retaining said sample (a);

means for retaining said solution (b) containing a known concentration of said tagged antigen; and

means for retaining said solution (c) of known concentration of said antibody; and

pumping means for withdrawing and mixing liquid portions from each of said retaining means to form said sample mixture and for propelling said sample solution into said time controlled incubator means.

6. The apparatus of claim 5, wherein said means for retaining the sample (a) is a disc, said disc holding a plurality of sequentially placed cups, and wherein said disc is rotatable so that each of said cups can sequentially be brought to a feeding position, and wherein a pipette, coupled to said pumping means, is situated at said feeding position, said pipette being movable between a first position wherein said pipette is dipped into said cup brought to said feeding position, and a second position wherein said pipette is incapable of receiving a sample, but which does not hinder the rotation of said disc, wherein as each sequential sample is desired to be introduced into the system, said disc is rotated to said feeding position for the next sequentially placed cup and said pipette is brought to its feeding position wherein a sample from the cup is extracted into said pipette as a result of a vacuum created in said pipette by said pumping means.

7. The apparatus of claim 6, wherein said disc holds a plurality of sequentially placed series of cups, each series comprising a cup containing said sample to be assayed, a cup containing said solution of tagged antigen and a cup containing said solution of antibody, and wherein said disc is rotatable so that each of said series is brought to said feeding position, and wherein an equal number of pipettes as the number of cups in said series is provided, each being coupled to said pumping means, such that after the disc is rotated to said feeding position, each of said pipettes is brought into one of the cups of said series so that feeding from the series occurs simultaneously, and wherein the pipetted solutions and samples are mixed and fed to said time controlled incubation means by said pumping means.

8. The apparatus of claim 1, wherein said time controlled incubation means is a cup wherein the said sample solution is deposited for a predetermined period of time before being introduced into said radioactivity detector means by said introduction means.

9. The apparatus of claim 1, wherein said introduction means is a temperature controlled conduit through which the sample solution is passed which is of a length such that as the solution is passed therethrough at a predetermined speed controlled by said pumping means, incubation is sufficiently completed.

10. The apparatus of claim 9, wherein a multiplicity of sample solutions to be assayed are introduced sequentially into said conduit such that incubation of said multiplicity of samples occurs simultaneously and in sequence.

11. The apparatus of claim 10, wherein each of said samples are separated by introduction of a buffer solution between each successive sample, wherein said buffer solution is fed to said conduit by a buffer feed means.

12. The apparatus of claim 5, wherein said pumping means is a peristaltic pump.

13. The apparatus of claim 12, which further comprises bubbler means positioned between said pumping means and said incubation means for intermittently injecting bubbles of air into said liquid steam emanating from said pumping means in order to provide a more uniform flow of liquid through said incubation means.

14. The apparatus of claim 13, which further comprises means for removal of bubbles from said sample after said sample passes said incubation means.

15. The apparatus of claim 1, wherein said isolation means comprises a valve which is rotatable between first and second positions, and which connects a conduit from said incubation means with a conduit to said radioactivity detector means, and with a conduit to waste, wherein in said first position, said conduit connects with said conduit to said waste, and wherein a pressure control means is provided such that the pressure within said conduit for said incubation means is maintained relatively constant irrespective of whether said valve is in its first or second positions.

16. The apparatus of claim 1, wherein said radioactivity detector means is a gamma detector.

17. The apparatus of claim 3, wherein said second section comprises a cup for retention of a solution, wherein said cup has inlet and outlet ports situated at the bottom thereof to permit filling and evacuation of said cup with minimal splashing of solution onto the walls thereof.

18. The apparatus of claim 1, wherein said separating means is a packed, continuous flow column containing an adsorbent which selectively adsorbs antigen which has not been reacted with antibody during said incubation.

19. The apparatus of claim 18, wherein said adsorbent is separated into a first zone which is packed with charcoal and a second zone which is packed with an ion-exchange resin.

20. The apparatus of claim 1, wherein said separating means is a continuous flow column containing an adsorbent which selectively adsorbs said antigen-antibody reaction complex formed during said incubation.

21. The apparatus of claim 1, wherein said means for increasing the rate of flow of the said incubated sample is a means for introducing a relatively high flow rate of buffer solution into the conduit carrying the incubated sample which moves the sample into, and through, the said separating means, and which simultaneously moves tailings into said separating means.

22. A method for continuous radioimmunoassay, which comprises:

incubating for a predetermined time period, a multiplicity of sample solutions, each of which comprises a mixture of (a) a sample possibly containing an antigen intended to be assayed, (b) a solution of a known concentration of an antigen tagged with a radioactive isotope, and (c) a solution of a known titer of antisera containing antibodies reactable with said antigen, the concentration of said antibody being selected so as to form an incubated sample containing an antibody-antigen complex, and containing unreacted antigen and tagged antigen, if said sample contains an antigen;

directing said incubated sample into a radioactivity detector which is capable of providing a kinetic measurement of radioactivity as the solution is continuously passed therethrough;

comparing the amount of radioactivity in said concentrated sample with a predetermined threshold and activating a timing means when said threshold is exceeded;

isolating said incubated sample from any additional samples being incubated on a predetermined signal from said timing means;

separating said incubated solution into a first portion containing unreacted antigen and tagged antigen and a second portion containing antibody-antigen and antibody-tagged antigen complexes;

directing one of said portions into a radioactivity detector, detecting the amount of radioactivity in said portion while said portion is maintained in a virtually static condition, and recording said measurement;

evacuating said portion to waste;

directing a subsequent incubated sample into said radioactivity detector on a second predetermined signal from said timing means so that the next subsequent incubated sample to be assayed arrives in the radioactivity detector for the next kinetic measurement after the previous static radioactivity measurement is completed; and

comparing the ratio of the static radioactivity measurement to the kinetic measurement with a precalibrated ratio to determine the quantity of antigen being assayed.

23. The continuous method of claim 22, wherein said incubated sample is isolated, the flow rate of the incubated sample through a separation means is increased by a means activatable by a predetermined signal from said timing means.

24. The continuous method of claim 22, wherein said sample solution is passed in laminar flow through a conduit maintained at controlled incubation temperature, and wherein bubbles are intermittently introduced into the said conduit to assure uniform movement of the sample solution through the conduit, and wherein the bubbles are released at the termination of the incubation procedure.

25. The continuous method of claim 22, wherein said incubated sample is directed through a conduit through an isolation valve which isolates the sample from any additional incubating samples, and into the said radioactivity detector, wherein the conduit leading to the isolating valve is maintained at selected constant pressure.

26. The continuous method of claim 22, wherein the separation of the first and second portions is effected in an adsorption column wherein the antigen and isotope tagged antigen are adsorbed and the antigen-antibody complex and tagged antigen-antibody complex are permitted to flow therethrough.

27. The continuous method of claim 26, wherein said adsorption column is a combination of a first section which is packed with charcoal and a second section which is packed with Dowex 1 anion exchange resin.

28. The method of claim 26, wherein the column adsorbent is charcoal.

29. The method of claim 26, wherein the column material is a Sephadex type molecular sieve.

30. The continuous method of claim 23, wherein the flow rate of the incubated sample through the separation means is increased by injecting a high flow rate stream of buffer solution into the said conduit leading to the radioactivity detector.

31. The continuous method of claim 24, wherein said multiplicity of sample solutions form a continuus stream wherein each sample solution is isolated by portions of a buffer solution which is introduced between each sample solution within said incubation conduit.

32. The continuous method of claim 22, wherein sequential sample solutions being assayed contain different antibody-antigen systems.

33. The continuous method of claim 32, wherein sequential sample solutions being assayed contain the same sample from the same sample source, but contain different antibody and tagged antigen solutions so that sequential assays are accomplished for different antigen contents for the same patient sample.