Automated analysis equipment and assay method for detecting cell surface protein and/or cytoplasmic receptor function using same

• US 5,670,113 A
• Filed: 06/20/1994
• Issued: 09/23/1997
• Est. Priority Date: 12/20/1991
• Status: Expired due to Term

• Current Assignee: Merck Sharp & Dohme Corporation (Merck & Co., Inc.)
• Original Assignee: Sibia Neurosciences, Inc. (Merck & Co., Inc.)
• Inventors: Velicelebi, Gonul, Brust, Paul, Akong, Michael Anthony, Harpold, Michael Miller
• Primary Examiner(s): PYON, HAROLD Y
• Application Number:
  US08/244,985
• Time in Patent Office: 1,191 Days
• Field of Search: 422/62, 422/63, 422/64, 422/65, 422/82.08, 422/82.09, 422/67, 422/82.05, 436/43, 436/50, 436/164, 356/402, 356/414, 356/416, 356/418, 356/432, 356/436
• US Class Current: 422/63
• CPC Class Codes: G01N 2035/042 moved independently, e.g. by fork manipulator
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 2333/705 Assays involving receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5058 Neurological cells
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels
• CPC Class Codes: G01N 35/028 having reaction cells in the form of microtitration plates
• CPC Class Codes: G01N 35/109 with two horizontal degrees of freedom
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/809 Multifield plates or multicontainer arrays
• CPC Class Codes: Y10T 436/11 Automated chemical analysis
• CPC Class Codes: Y10T 436/115831 Condition or time responsive

Abstract

Automated measurement apparatus and methods for automated drug screening and for studying ion channel and cell surface receptor activity are provided. The apparatus is designed to initiate and measure rapid or transient events, such as cell receptor and/or ion channel activity. The apparatus can effect measurements of transient reactions in or more samples in a multi-well container, initiate the reaction with reagent addition and measure a resulting attribute of the sample for a period of time. The apparatus is capable of substantially continuously measuring and recording data corresponding to the measured attribute before, during and after initiation of the reaction so that a time course of the rapid or transient event is determined. Automated drug screening methods and methods for studying receptor and ion channel activity using the apparatus are also provided. The drug screening assays identify compounds that activate, inhibit or potentiate cellular ion channel or receptor activity.

Claims

1. In a computer-controlled fluorescence-measuring apparatus comprising means for measuring the fluorescence of individual samples contained in a plurality of wells of a multi-well plate, a method for measuring transient reactions comprising:

(a) identifying a predetermined one of the plurality of wells containing a sample to be measured;

(b) moving the predetermined well by the apparatus to a reagent addition position of the apparatus;

(c) adding reagent by the apparatus to the predetermined well at the reagent addition position;

(d) moving the predetermined well to a measurement position of the apparatus after the addition of the reagent; and

(e) measuring the post-reagent addition fluorescence value of the solution in the predetermined well at the measurement position after the addition of the reagent to the predetermined well, wherein;

the movement of the predetermined well, step (d), and the commencement of measurement, step (e), occurs within a predetermined time period that is less than the time during which a reaction that is detected by the fluorescence reaches its peak, and measurement is effected for a predetermined amount of time;

(f) moving the predetermined well by the apparatus to a fluorescence measurement position prior to the adding reagent step;

(g) measuring the pre-reagent fluorescence value of the solution in the predetermined well at the measurement position prior to the addition of the reagent; and

(h) recording the pre-reagent fluorescence value measured.

2. The method of claim 1, further comprising the step of:

(i) identifying different ones of the plurality of wells, one at a time, to be the predetermined well and repetitively performing steps (b) through (f) for each well identified as the predetermined well.

3. The method of claim 1, further comprising:

(j) identifying different ones of the plurality of wells, one at a time, to be the predetermined well and performing steps (b) through (h) for each well identified as the predetermined well.

4. The method of claim 1, further comprising, optically irradiating the sample in the predetermined well prior to commencement of measurement.

5. The method of claim 1, wherein the predetermined time period is less than or equal to about 10 seconds.

6. In a computer-controlled fluorescence-measuring apparatus comprising means for measuring the fluorescence of individual samples contained in a plurality of wells of a multi-well plate, a method for measuring transient reactions comprising:

(a) identifying a predetermined one of the plurality of wells containing a sample to be measured;

(b) moving the predetermined well by the apparatus to a reagent addition position of the apparatus;

(c) adding reagent by the apparatus to the predetermined well at the reagent addition position;

(d) moving the predetermined well to a measurement position of the apparatus after the addition of the reagent; and

(e) measuring the post-reagent addition fluorescence value of the solution in the predetermined well at the measurement position after the addition of the reagent to the predetermined well, wherein;

the movement of the predetermined well, step (d), and the commencement of measurement, step (e), occurs within a predetermined time period that is less than the time during which a reaction that is detected by the fluorescence reaches its peak, and measurement is effected for a predetermined amount of time;

(f) recording the fluorescence value measured in the post-reagent measuring step.

7. The method of claim 6, further comprising the step of:

(j) identifying different ones of the plurality of wells, one at a time, to be the predetermined well and repetitively performing steps (b) through (f) for each well identified as the predetermined well.

8. A method of claim 6, wherein the measuring step comprises optically irradiating the sample in the predetermined well.

9. The method of claim 6, wherein the predetermined time period is less than or equal to about 10 seconds.

10. The method of claim 6, further comprising:

(g) moving the predetermined well by the apparatus to a fluorescence measurement position prior to the adding reagent step;

(h) measuring the pre-reagent fluorescence value of the solution in the predetermined well at the measurement position prior to the addition of the reagent; and

(i) recording the pre-reagent fluorescence value measured.

11. The method of claim 10, further comprising:

(k) identifying different ones of the plurality of wells, one at a time, to be the predetermined well and performing steps (b) through (i) for each well identified as the predetermined well.

12. An automated measurement apparatus, comprising:

control means for coordinating the operation of the apparatus;

sample-containing means comprising a plurality of wells for containing individual solution samples;

means responsive to the control means for aligning one or more predetermined wells of the plurality of wells with a predetermined position;

means responsive to the control means for adding reagent to the one or more predetermined wells while aligned with the predetermined position; and

measurement means responsive to the control means for;

(i) measuring an attribute of each sample in the one or more of the predetermined wells while aligned with the predetermined position; and

(ii) measuring the attribute of each sample in the predetermined wells so that all wells are measured simultaneously or so that one well at a time is measured, wherein;

the measurement means commence measurement within a predetermined time period after reagent addition that is less than the time required for the transient reaction to reach its peak response; and

the measuring means is also adapted to measure the attribute of each of the samples in the predetermined position before and during the addition of reagent to the sample by the reagent adding means.

13. The apparatus of claim 12, wherein the measuring means comprise means for optically irradiating a sample in a well.

14. The apparatus of claim 12, wherein the measuring means comprises means for periodically measuring the attribute of each of the samples in the predetermined position for a predetermined time period beginning before the reagent addition and ending after the reagent addition and the apparatus comprises means for storing representations of the attribute measured for each of the samples in the predetermined position at each periodic measurement and means for storing indicia of a beginning and an ending of reagent addition.

15. The apparatus of claim 12, wherein the measuring means optically measures the attribute of the samples, the sample containing means is fabricated from material which is substantially optically transparent in a range of wavelengths at which the sample is optically measured and the measuring means is disposed in a position to measure the attribute of the samples through at least a portion of the sample containing means.

16. The apparatus of claim 12, wherein the reagent adding means is disposed above the predetermined ones of the wells and the measuring means is disposed beneath the predetermined ones of the wells when the predetermined ones of the wells are in the predetermined position.

17. The apparatus of claim 12, comprising means for optically exciting the samples in the predetermined position and the measuring means comprises means for optically measuring the response of the samples in the predetermined ones of the wells to the optical excitation.

18. The apparatus of claim 12, comprising means responsive to the control means for moving the reagent adding means from the predetermined position to a reagent source position.

19. The apparatus of claim 12, comprising means responsive to the control means for washing at least one of the wells.

20. The apparatus of claim 12, wherein the attribute is fluorescence and the means for measuring comprise means for measuring the fluorescence of the sample.

21. The apparatus of claim 20, further comprising means for recording the fluorescence magnitude value.

22. The apparatus of claim 12, wherein the means for optically exciting comprises means for alternatingly optically exciting the samples in the predetermined positions with radiation centered at a first and a second optical wavelength;

and

the means for measuring comprises means for measuring a response of the samples in the predetermined position to excitation by each of the first and the second excitation wavelengths.

23. The apparatus of claim 22, comprising means for computing a ratio of the measured response of each of the samples in the predetermined position to optical excitation at the first and the second wavelengths.

24. The apparatus of claim 12, wherein the control means comprise computer means.

25. The apparatus of claim 24, further comprising input means for inputting instructions to the computer.

26. The apparatus of claim 24, wherein the computer means generates plate movement commands and the moving means responds to the plate movement commands by moving the predetermined well(s) to the reagent adding position and to the measurement position.

27. The apparatus of claim 24, wherein the computer means generates a reagent pump command when the predetermined well(s) is at a reagent adding position and the reagent adding means responds to the reagent pump command by adding reagent to the predetermined well(s).