Automated system for performing fluorescent immunoassays

Claims

We claim:

1. A fluorescence immunoassay system for sequentially quantitating relatively small amounts of a clinically significant composition in a multiplicity of independent liquid samples, the samples including fluorescent particles, comprising:

transparent means defining a hollow cell for holding the sample;

a light source for generating a stable light beam directed onto the sample;

whereby the light beam causes fluorescent emissions by the particles in the sample, the intensity of the emissions being a function of the intensity of the light beam and the quantity of fluorescent particles in the sample;

means in optical communication with the transparent means for detecting photons resulting from fluorescent emissions by the particles when excited by the light beam;

whereby the number of photons detected by the photon detecting means is a function of the number of fluorescent particles in the sample;

sampler means for sequentially aspirating a liquid from a multiplicity of upwardly open vials, each vial holding one sample;

means for flowing the aspirated liquid to the cell; and

means for agitating the sample in the vial prior to aspirating the sample from the vial to ensure a uniform distribution of fluorescent particles in the aspirated liquid.

2. System according to claim 1 wherein the hollow cell has a plurality of mutually perpendicular outer surfaces, one of the surfaces facing the light source and being perpendicular to the light beam.

3. System according to claim 1 including a power supply for the light source, means for sensing the intensity of the beam, and circuit means operatively coupled with the sensing means and the power supply for adjusting the output of the power supply in response to changes in the intensity of the light beam so that the light beam intensity remains substantially constant.

4. System according to claim 3 wherein the sensing means comprises a photodiode, and wherein the circuit means includes means for amplifying an output of the photodiode, means for comparing the amplified output of the photodiode with a reference signal to generate a difference signal, and means for applying the difference signal to the power supply to correspondingly adjust its output to the light source.

5. System according to claim 4 wherein the light source comprises a tungsten-halogen incandescent lamp and the photodiode comprises a silicon photodiode.

6. System according to claim 4 including filter means between the light source and the sample for conditioning the light beam so as to minimize light scattering and enhance fluorescent emissions by the particles, and wherein the photodiode is positioned relatively proximate to the transparent means and optically downstream of the filter means.

7. System according to claim 1 wherein the flowing means comprises an inlet to and an outlet from the cell, wherein the sampler means includes an aspirator for withdrawing the liquid samples from the sample vials, and further including intake tubing fluidly communicating the aspirator with the inlet; discharge tubing fluidly communicating the outlet with a sample discharge point, and a pump located downstream of the outlet for intermittently flowing a sample from the aspirator to the cell, for maintaining the sample in the cell for a period of time, and for thereafter removing the entire sample from the cell and the intake tubing and replacing the removed sample with a fresh sample; whereby the sample is not subjected to the flow inducing action of the pump until after it has passed the cell.

8. System according to claim 7 wherein the sampler means includes a container holding a rinsing solution, and including means for alternatively fluidly communicating the aspirator with the vials and the container.

9. System according to claim 8 including means for alternatingly fluidly communicating the aspirator with the vial and the container in response to each operation of the pump so that at least the intake tubing and the cell is cleaned with the rinsing solution prior to the replacement of the sample with the fresh sample.

10. System according to claim 1 wherein the photon detecting means includes means for generating signal pulses for individually sennsed photons, and further including digital pulse counting means having an input in communication with the signal pulse generating means for producing a numeric code representative of the number of pulses received at the input.

11. System according to claim 10 wherein the pulse counting means includes an enable input, and further including processor means coupled to the pulse counting means, the processor means having means for receiving and storing the numeric code from the pulse counting means, the processor means also having means for enabling the pulse counting means for a predetermined interval of time so that the numeric code stored is representative of concentration of the clinically significant compound.

12. System according to claim 11 wherein the processor includes means for storing the known concentrations of the clinically significant compound for a plurality of standard samples, means for storing a corresponding plurality of numeric codes corresponding to the measured fluorescent activity of the plurality of samples having known concentration, and arithmetic means for computing a calibration curve on the basis of the plurality of known concentrations and the plurality of numeric codes.

13. System according to claim 12 wherein said arithmetic means includes means for computing a calibration curve according to at least two mathematical hypotheses, and for choosing the mathematical hypothesis that provides the highest correlation coefficient.

14. System according to claim 1 wherein the photon detecting means includes means for generating signal pulses for individually sensed photons, and further comprising:

digital pulse counting means for receiving the signal pulses from said photon detecting means and for providing a numeric code output representative of the number of pulses counted; and

programmed microcomputer means in communication with the pulse counting means, the programmed microcomputer means having means for selectively enabling the counting means for a predetermined time interval such that the numeric code from the counting means is representative of the concentration of the clinically significant compound, the microcomputer means having means for storing known concentration information for a plurality of standard samples and means for accumulating and storing count information corresponding to said plurality of standard samples, the microcomputer having means for determining a calibration curve on the basis of the known concentrations and count information according to at least two mathematical hypotheses and for selecting the calibration curve that provides the best correlation coefficient.

15. System according to claim 1 wherein the sampler means comprises:

an aspirator including an upright, downwardly opening suction tube for sequential insertion into the vials;

tray means supporting the vials for incrementally moving the vials to an aspiration station;

positioning means for aligning the suction tube and the vial at the aspiration station; and

immersion means for vertically moving the suction tube into and out of a vial at the station so as to immerse a lower end of the tube in the sample in such vial.

16. System according to claim 15 wherein the agitating means comprises a rotary mixer carried by the aspirator.

17. System according to claim 16 wherein the mixer comprises a tubular member concentric with the suction tube, means for rotating the tubular member about its axis, and means for maintaining the suction tube stationary.

18. System according to claim 19 wherein the suction tube is disposed within the tubular member.

19. System according to claim 18 wherein the aspirator includes a frame, wherein the suction tube is fixedly mounted to the frame, and including means for rotatably mounting the tubular member to the frame.

20. System according to claim 19 wherein the tubular member has a lower end protruding past a corresponding end of the suction tube.

21. System according to claim 20 wherein the lower end of the tubular member is defined by a cylindrical wall, and including at least one downwardly opening groove in the cylindrical wall to facilitate the agitation of the liquid in the vial.

22. System according to claim 15 wherein the immersion means comprises an upright rod having an upper end to which the aspirator is mounted, and means for vertically reciprocating the rod so that the lower end of the suction tube is above an upper end of the vial at the aspiration station when the rod is in its raised position and immersed in the sample and proximate but spaced from a bottom of the vial when the rod is in its lowered position.

23. System according to claim 22 including first drive means for vertically reciprocating the rod comprising a rack and pinion drive including a load reversible electric motor for rotating the pinion of the drive and for reversing its rotational direction whenever the rod encounters a torque of a predetermined magnitude which opposes the continued movement of the rod in a given direction; whereby misalignments and an interference between the suction tube and objects including the vial at intermediate positions of the reciprocating rod automatically terminates further movement of the rod and of the suction tube in such direction and thereby prevents damage to the apparatus and the vial.

24. System according to claim 23 including optical limit switches operatively electrically coupled with the electric motor for sensing raised and lowered positions of the rod and for de-energizing the electric motor in response to sensing the presence of the rod in either one of said positions to prevent further travel of the rod in a given direction.

25. System according to claim 15 including a container holding a rinsing solution for rinsing the interior and the exterior of the suction tube after the sample in the vial has been withdrawn to prevent contamination of the sample in the next vial to be aspirated by the sample from the previously aspirated vial, wherein the positioning means includes means for aligning the suction tube with the container, and wherein the immersion means includes means for vertically moving the suction tube into and out of the container when the container and the tube are in alignment.

26. System according to claim 25 wherein the tray means includes a tray having means for holding the vials, and means for moving the tray along a predetermined path past the aspiration station, and wherein the positioning means includes means for arresting movement of the tray means when a vial is at the aspiration station.

27. System according to claim 26 wherein the tray means includes means for rotating the tray about an axis and means for mounting the vials to the tray in a general circular pattern concentric with respect to the tray axis, and wherein the tray moving means comprises means for rotatably moving the tray in fixed increments about its axis so as to present a vial at the aspiration station after an incremental movement of the tray.

28. System according to claim 27 wherein the vial holding means includes means for arranging the vials in a plurality of concentric, radially spaced rows; and wherein the positioning means includes means for moving the suction tube in a direction transverse to the direction of movement of the vials past the aspiration station into alignment with a plurality of vials which equals the plurality of rows while the tray remains stationary.

29. System according to claim 28 wherein the means for moving the suction tube comprises a frame for mounting the tube and means for pivoting the frame about an aspirator axis parallel to the suction tube between a first position in which the tube is aligned with a vial at the station in a first row and a second position in which the tube is in alignment with a vial at the station in the second row.

30. System according to claim 29 wherein the vial mounting means includes means arranging the vials at the aspiration station so that centers of such vials substantially lie on a circularly arcuate line having as its origin the aspirator axis.

31. System according to claims 30 wherein the container is positioned on the arcuate line, and wherein the means for aligning the suction tube with the container comprises means for pivotally moving the frame about the aspirator axis until the suction tube is in substantial alignment with the container.

32. System according to claim 31 wherein the positioning means includes second drive means for pivotally moving the frame about the aspirator axis, the second drive means including signal means for indicating when the tube is in alignment with any one of the container and the vials at the aspiration station, and means responsive to the signal means for arresting the pivotal movement of the frame when the suction tube is in alignment with a vial or the container into which the suction tube is to be moved.

33. System according to claim 32 wherein the immersion means includes first drive means having an upright rod connected with the aspirator, the rod being aligned with and mounted to rotate about the aspirator axis; and wherein the second drive means includes a crank drive operatively coupled with the rod and having a drive wheel, the crank drive being arranged so that one rotation of the drive wheel causes a pivotal movement of the frame and therewith of the suction tube from a given point along said arcuate line over all other points on said line and back to said given point.

34. System according to claim 15 wherein the tray means includes vial holding means arranging the vials in at least one circular row, means mounting the vial holding means for rotation about an upright tray axis so that rotation of the vial holding means sequentially presents the vials at the aspiration station, and a notched index wheel fixedly connected with the vial holding means and rotatable therewith, the index wheel having a number of notches corresponding to the number of positions of the vial holding means at which a vial is presented at the aspiration station, and wherein the positioning means comprises a detent, and spring means resiliently biasing the detent into engagement with the notches, the detent being positioned so that a vial is aligned with the suction tube at the aspiration station when the detent fully rests in a corresponding notch in the index wheel.

35. System according to claim 34 wherein the tray means includes third drive means operatively engaging the index wheel for incrementally advancing the index wheel to sequentially engage the notches on the index wheel with the detent.

36. System according to claim 35 wherein the notches in the index wheel are radially oriented, and wherein the detent is biased towards the index wheel in a radially inward direction.

37. System according to claim 35 wherein the third drive means comprises drive pin means having a surface shaped to engage the notches in the index wheel and mounted adjacent of the index and means for moving the surface to engage a notch on the index wheel and rotatably advance the index wheel to engage the detent with another notch and to thereby align another vial with the suction tube at the aspiration station.

38. System according to claim 37 wherein the cam surface is eccentrically mounted to a rotatable cam wheel, and including means for resiliently biasing the surface towards the index wheel, and means limiting the extent to which the surface can move towards the index wheel so that the surface engages the index wheel during only a portion of the rotation of the cam wheel.

39. System according to claim 38 wherein the third drive means includes means for rotating the cam wheel through one full revolution for rotatably advancing the index wheel through an arc no greater than the arc between adjoining notches.

40. A system for performing fluorescence immunoassay on a multiplicity of individual liquid test samples including fluorescent particles and stored in a like multiplicity of relatively long, upright, upwardly open vials for quantitating relatively small amounts of a clinically significant composition comprising:

a vial holder holding the vials in an upright position and arranging the vials in at least one row;

advancing means for moving the vial holder to present each vial at an aspiration station; an aspirator including a frame, a vertically oriented suction tube fixedly mounted to the frame;

an upwardly open container spaced from the rows and located proximate the aspiration station for holding a volume of a rinsing solution;

positioning means for moving the frame to alternatively substantially vertically align the suction tube with a vial at the aspiration station and with the container;

immersion means for raising and lowering a lower end of the suction tube between a raised position at which the lower suction tube end clears an upper end of the vial at the aspiration station and the container and a lowered position at which the lower suction tube end is immersed in liquid in the tube or the container;

means for operating the positioning means and the immersion means so as to immerse the suction tube in the container after each immersion of the suction tube in a vial at the aspiration station;

a transparent housing defining an interior cell and an inlet and an outlet to and from the cell, respectively;

conduit means including pump means fluidly communicating the suction tube with the cell inlet for flowing a sample from the tube to the cell;

means for holding a portion of each sample for at least a minimum period of time stationarily in the cell;

optical means including a light source generating a light beam which is directed into the cell for causing fluorescent emissions by the particles in the cell, the intensity of which is a function of the intensity of the light beam and the quantity of particles in the cell;

photosensing means for sensing the fluorescent emissions over a predetermined time period and for generating corresponding output signals;

means for stabilizing the intensity of the light beam to prevent substantial variations in the intensity of the light beam from a preset light beam intensity;

discriminator means for eliminating from the output signals at least a substantial portion of any background noise signals which are included in the output signals;

digital counting means responsive to the output signals; and

microcomputer means including

means for receiving and storing numeric information from the counting means,

means for enabling the counting means for a predetermined length of time so that the numeric information is representative of the number of particles in the cell,

means for enabling the pump activating means at a time when the counting means is not enabled so that counting may occur while the sample is stationary in the cell,

means for storing known concentration information for a plurality of standard samples and means for accumulating and storing count information corresponding to said plurality of standard samples, and

means for determining a calibration curve on the basis of the stored known concentrations and count information according to at least two mathematical hypotheses and for selecting the calibration curve that provides the best correlation coefficient.

41. System according to claim 40 including means defining a generally L-shaped optical chamber having perpendicular optical axes; and wherein the cell is disposed at the intersection of the axes.

42. System according to claim 40 wherein the optical means and the photosensing means are disposed in the optical housing and aligned with the first and second optical axes, respectively.

43. System according to claim 42 wherein the transparent housing has a generally square cross-section, the housing including first and second perpendicular sides which are arranged perpendicular to the first and second optical axes, respectively.

44. System according to claim 43 wherein the cell has a square cross-section and includes first and second perpendicular walls which are parallel to the first and second sides of the housing.

45. System according to claim 40 wherein the photosensing means comprises a photomultiplier tube, wherein the output signals comprise signal pulses generated by photons from fluorescent emissions of the particles in the cell and noise pulses; and wherein the discriminator means includes means for eliminating the noise pulses from the output signals before the signal pulses are fed to the processing means.

46. System according to claim 45 wherein the signal pulses have an amplitude greater than the noise pulses, and wherein the discriminator means includes an amplitude discriminator for eliminating the noise pulses from the output signals.

47. System according to claim 40 including indexing means for positioning the vial at the aspiration station, the indexing means being independent of the advancing means.

48. System according to claim 40 wherein the vial holder arranges the vials in at least two side by side, parallel rows; wherein the frame moving means includes means for pivoting the frame about an upright frame axis so that the suction tube moves along a circularly arcuate path; and wherein a vial in each row is simultaneously positioned at the aspiration station.

49. System according to claim 40 wherein the vial holder arranges the vials in a plurality of rows; and including means for aligning a vial in each row with the aspiration station, the aligning means comprising an index member fixedly attached to the vial holder for movement therewith, a detent biased towards the indexing member, the indexing member and the detent defining cooperating concave and convex index surfaces, the surfaces being arranged so that upon the mutual engagement of the detent with a corresponding surface on the indexing member a vial in each row is aligned with the aspiration station, the number of surfaces on the indexing means being equal to the number of vials held by the holding means divided by the number of rows.

50. A system for performing fluorescent immunoassay on a multiplicity of individual liquid test samples including fluorescent particles and stored in a like multiplicity of vials for quantitating relatively small amounts of a clinically significant composition comprising: a transparent housing having a square cross-section and including an interior sample cell of a square cross-section, corresponding housing sides and cell walls being parallel to each other, the housing including an inlet to and an outlet from the cell; an intake conduit in fluid communication with the inlet and having an other end; a sampler including an aspirator, a container for holding a rinsing solution and means supporting the vials; means for alternatively aligning the aspirator with a vial or with the container; an intake conduit fluidly communicating the aspirator with the cell inlet; a discharge conduit in fluid communication with the outlet; pump means disposed downstream of the outlet and cooperating with the discharge conduit for flowing a sample or rinsing solution by suction through the intake conduit, the cell and a portion of the discharge conduit and for flowing it to a point of discharge; sequencing means for intermittently activating the pump means and for alternatingly immersing the aspirator in the container and in a vial to alternatingly flow a sample or rinsing solution into the cell; the sequencing means including means for activating the pump means for a sufficient length of time to evacuate the entire liquid in the cell and replace it with fresh liquid from one of the container or one of the vials; an optical housing defining a pair of perpendicular, optical branches defining perpendicular optical axes, the cell being disposed in the optical housing and at an intersection of the axes, a light source in one of the branches for generating a light beam and first optical means disposed in the optical branch between the light source and the cell for removing from the light beam substantially all light other than light of a wavelength which causes the fluorescent particles to emit fluorescent emissions; a photomultiplier in the other branch and second optical means for directing the fluorescent emissions onto the photomultiplier, the photomultiplier generating a signal pulse for each fluorescent transmission photon received thereby and noise signals; the housing including means for preventing light other than fluorescent emissions caused by the direct excitation of the fluorescent particles by the light beam from reaching the photomultiplier;

light beam stabilization means operatively coupled with the light source and including a photosensor disposed in the optical housing proximate the transparent housing and optically downstream of the first optical means for sensing the light beam striking the housing and for adjusting its intensity so that the light beam remains substantially constant;

discriminator means operatively coupled with the photomultiplier for removing the noise signals and for generating output signals comprising substantially only signal pulses;

counting means operatively coupled with the discriminator means for counting the number of signal pulses emitted by the photomultiplier; and

microcomputer means having means for enabling the counting means over a predetermined, constant length of time and for forming an output which is an indication of the number of fluorescent particles in the sample cell, the microcomputer means having means operativly coupled with the pump means for deactivating the pump means during at least the predetermined length of time and for activating the pump means during other times so that a tested sample fluid from the cell is removed, the cell is rinsed with rinsing solution, and thereafter a fresh sample is flowed into the cell.

51. A system for performing fluorescent immunoassay on a multiplicity of individual test samples including fluorescent particles and stored in a like multiplicity of vials for quantitating relatively small amounts of a clinically significant composition, the system comprising: a vial holder arranging the vials in at least one row of a predetermined shape and maintaining the vials in an upright position so as to render them accessible from the top; means for intermittently moving the holder parallel to the row to sequentially position the vials at an aspiration station; a container located proximate the aspiration station for holding a rinsing solution; an aspirator frame including at least a portion disposed above uppermost ends of the vials at the aspiration station and an uppermost end of the container; a suction tube affixed to the frame and having an open lower end; flow means in fluid communication with the suction tube for subjecting the tube to a vacuum to draw a liquid through the tube when the lower tube end is immersed in a liquid; a rotary mixer secured to the frame proximate the tube and depending from the tube to a point below the lower tube end, the tube and the mixer being constructed so that they can be simultaneously inserted in a vial; immersion means for reciprocating the frame in a vertical direction over a sufficient distance so that in a raised position of the frame the lower end of the mixer is above the vials and the container and in a lowered position of the frame the lower end of the mixer and the lower end of the tube are immersed in liquid in the vial or the container; positioning means for moving the frame along a predetermined path to alternatively substantially align the tube and the mixer with a vial at the aspiration station or with the container; a transparent housing defining a sample cell having an inlet in fluid communication with the flow means and the suction tube and an outlet; a light source for generating a stable light beam focused on the cell; whereby the light beam causes fluorescent emissions by particles in the cell, the intensity of the emissions being a function of the intensity of the light beam and the quantity of fluorescent particles in the cell; means in optical communication with the transparent housing for detecting photons resulting from fluorescent emissions by the particles when excited by the light beam; whereby the number of photons detected by the photon detecting means is a function of the number of fluorescent particles in the cell; and means for sequentially energizing the holder moving means, the positioning means, the immersion means, the mixer and the flow means to initially align the tube and the mixer with a vial at the aspiration station, to thereafter immerse the sample in the vial at the aspiration station, mix the sample with the mixer and thereafter withdraw at least a portion of the sample from the vial through the tube and flow it to the cell, thereafter withdraw the tube and the mixer in an upward direction from the vial and move them into registration with the container, immerse them in the container and flow rinsing solution from the container and through the suction tube until it at least fills the cell to remove from the tube, the mixer, the flow means and the cell substantially all previous sample remnants, and to thereafter realign the mixer and the tube with another vial at the aspiration station for withdrawing a fresh sample from said another vial; whereby the samples in the vials are uniformly mixed before their withdrawal therefrom and a cross-contamination between samples is prevented.

BACKGROUND OF THE INVENTION

The present invention relates to an automated system for the immunoassay of subnanogram quantities of certain compositions by molecular fluorescence.

The quantitative determination of small amounts of clinically significant compounds, such as metabolites, hormones, drugs and proteins are of recognized diagnostic importance. Radioimmunoassay (RIA) has become the standard method for making such determinations because of its sensitivity and specificity.

However, RIA has certain drawbacks. The radioactivity associated with RIA may present psychological or physical health hazards to the technologists, requires special licensing from nuclear regulatory agencies, requires the special disposal of wastes, limits the useful life of a reagent kit to a few months at the most, and requires relatively expensive instrumentation. To circumvent these drawbacks, alternative methods including fluorescence immunoassay (FIA) have been developed.

FIA is a technique in which a fluorescent molecule is substituted for the radioactive label used in RIA. Some of the advantages of FIA are: no radioactivity, a much longer useful lifetime of the test components or chemicals necessary for the assay, and relatively less expensive instrumentation for performing the assays.

By way of background, the commonly owned co-pending U.S. patent application bearing Ser. No. 875,475, filed Feb. 6, 1978 for SOLID PHASE IMMUNOFLUORESCENT ASSAY METHOD, now U.S. Pat. No. 4,201,763, describes in detail a FIA method for antigens (or haptens) and their antibodies through the use of an immune reactant related to the antibody or antigens to be determined which is covalently bonded or coupled to polymeric particles whose size permits direct measurement of a labelled immunological reagent's fluorescence in an aqueous suspension thereof by direct optical spectroscopy. A key to the method described in that U.S. patent application lies in the selection of certain types of polymeric particles in sizes which provide a substantially homogeneous suspension during execution of the assay. It has been discovered that such a condition exists and that direct fluorometric measurements can be made when the polymeric particles have a size of about 0.1-10 microns.

Utilizing such particles, an appropriate immune reactant immunologically related to unknown antigen or antibody to be determined is covalently bonded thereto. The particles, unknown immune reactant, and appropriate fluorescently labelled immune reactant are mixed under conditions so that a quantity of the labelled immune rectant proportional to the concentration of the unknown immune reactant is immunologically bound, directly or indirectly, to the particles.

In accordance with said co-pending U.S. patent application, the FIA provides water insoluble hydrophilic polymeric particles of about 0.1-10 microns in size and having covalently bonded thereto the immunological homolog for an antigen or antibody to be determined. The particles are combined with the antigen or the antibody to be determined in an aqueous solution to form an immunological bond therebetween. A fluorescently labelled antigen or antibody corresponding to the antigen or antibody to be determined is immunologically bound to the particles.

Any suitable water insoluble polymeric particle may be utilized in the FIA described in said U.S. patent application. Generally, the particle will be in spherical or bead form and will be selected from polymers which can be derivatized to give a terminal primary amine, terminal carboxyl, or hydroxide group. The antibody or antigen is then immobilized on the particle under conventional reaction conditions to produce a covalent bond therebetween. Useful polymeric particles are formed, for example from crosslinked polyacrylamides. Other suitable polymeric particles are described in said U.S. patent application and in the references cited therein.

The particles are then physically separated, usually by centrifuging them, typically at 1500 g to pack the particles at the bottom of the test tube into a pallet. The supernatant is decanted, to the extent necessary the tube or vial is blotted dry and a barbital buffer is added to the pellet in the test tube to reconstitute it and resuspend the particles to form a suspension which includes the fluorescent particles.

The suspension is then analyzed on a fluorometer to determine the concentration of fluorescent particles in the sample to obtain information from which unknown antigen or antibody can be determined.

As has been customary in the past, these tests have heretofore been performed manually one after the other. This required, inter alia, a vigorous manual shaking of the test tube to reconstitute each pellet and resuspend the fluorescent particles. To obtain an accurate test it is, of course, necessary that the suspension be uniform which prolonged the time during which the tube had to be shaken. Thereafter, the sample was fluorometrically analyzed, either in the test tube or by pouring it from the tube into a suitable container of a fluorometer.

This procedure is time-consuming and requires the constant close supervision by a highly skilled technician. More importantly, it gives no assurance that an adequate mixing of the sample has taken place. Without such mixing, however, the ultimate readout is inaccurate and can render the entire test of questionable value. Further, the test is relatively expensive because of the close and constant supervision it requires.

A key to the success of FIA is the reliability and accuracy of the fluorometer over extended periods of time. In this regard, prior art fluorometers had certain shortcomings which could affect the ultimate readout and thus compromise the accuracy of the test. Conventional fluorometers that operate in an analog mode are unsatisfactory because of the relative insensitivity of such fluorometers when measuring the low light intensities encountered when performing FIA.

Better accuracy can be attained with photon-counting fluorometers which are relatively simple and inexpensive to construct. Robert E. Curry et al discuss the construction of photon-counting fluorometers (hereinafter "fluorometer" unless otherwise indicated) in "Design and Evaluation of a Filter Fluorometer that Incorporates a Photon-Counting Detector" on pages 1259-1264 of Clinical Chemistry, Vol. 19, No. 11, 1973, although the use of such fluorometers in conjunction with FIA has not heretofore been considered. The Article notes that photo-counting is an effective method for minimizing dark current contributions in photomultiplier tubes since electrons emitted from the dynodes are amplified less than electrons emitted from the photocathode and level discriminating circuitry can be used to differentiate between the dark current and photon signals.

For the determination of small amounts (i.e. from subnanomolar levels up) of clinically significant compounds by FIA, accuracy problems are, of course, not fully solved by employing a photo-counting fluorometer. Stray light, a non-uniform suspension of the fluorescent beads, light scattering, a variation in the magnitude of the samples' own fluorescence as well as changes in the primary light intensity all adversely affect the ultimate readout and lessen its accuracy. In addition, existing FIA methods must rely on an essentially manual, sample by sample determination of the fluorescence which requires the constant attention of highly skilled and, therefore, costly operations. This in turn has a tendency to drive up the already high costs for such tests.

SUMMARY OF THE INVENTION

The present invention provides an integrated system for conducting FIA on a large number of individual samples in an automatic, reliable, self-correcting and continuous manner for the quantitation of antigens or haptens and antibodies of any molecular weight and at concentrations from subnanomolar levels upwards. Initially, in one type of a competitive binding FIA, the antigen labeled with a fluorescent dye competes with the antigen in the sample or standard for a limited amount of antibody which is immobilized on a 0.1-10 micron polyacrylamide bead. After a suitable incubation, the labeled antigen bound to the antibody beads is separated from the free fluorescently-tagged antigen in the supernatant by centrifugation and decantation. After resuspending the antibody beads in buffer, the fluorescence bound to the beads is measured in accordance with the present invention in a sample analyzer that utilizes a feedback stabilized light source which illuminates a sample in a transparent holding cell to generate fluorescent emissions. The emissions are sensed by a photon-counting detector that forms photon generated output pulses from which background noise is effectively eliminated.

System electronics for the present invention employs large scale integration microcomputer architecture to provide an automated capability. In addition to supervisorial and sequencing tasks, the microprocessor performs data acquisition and data reduction operations to convert photon count information into antigen concentration. The apparatus of the present invention assures a measurement precision and accuracy of about one to three percent for the above indicated relatively low concentrations being measured.

The present invention also overcomes the disadvantages inherent in prior art procedures for reconstituting the pellets in the bottom of test tubes and especially for uniformly mixing the pellets with the buffer solution and for presenting the resulting suspension to a fluorometer so that it can be appropriately analyzed. The present invention accomplishes this by fully automating both the mixing and the withdrawal of the suspension from the vials so that they can be presented to the appropriate instrument such as a fluorometer.

The apparatus of the present invention is fully automated, being capable of processing one sample after the other on a continuing basis. For this purpose the analyzer includes a sample cell which is fluidly communicated with a suitable pump that transports a predetermined sample volume into the cell, maintains the volume in the cell until its fluorescence has been measured, and thereafter replaces the sample in the cell with a new one. The pump is operatively coupled with an automatic sample mixing and retrieving unit, hereinafter sometimes referred to as "sampler".

Generally speaking, the analyzer of the present inv