EP0016165A1

EP0016165A1 - Method of conducting radioassay using a combined reaction/filter vial

Info

Publication number: EP0016165A1
Application number: EP79900917A
Authority: EP
Inventors: William W. Miller
Original assignee: Beckman Instruments Inc
Current assignee: Beckman Coulter Inc
Priority date: 1978-07-25
Filing date: 1980-03-11
Publication date: 1980-10-01
Also published as: JPS55500529A; WO1980000371A1; EP0016165A4

Abstract

Une methode d'essai immunologique par mesure de radioactivite en phase solide (RIA) utilise une fiole combinee de reaction/filtration comptage (10) a chaque etape de l'essai. La reaction s'effectue dans la fiole (10), le filtre de la fiole (16) est employe pour arreter un precipite marque derive de la reaction, et la fiole (10) est placee dans un compteur nucleaire pour mesurer le precipite marque.A solid phase radioactive activity (RIA) immunoassay method uses a combined reaction / filtration counting flask (10) at each stage of the assay. The reaction is carried out in the flask (10), the filter in the flask (16) is used to stop a marked precipitate derived from the reaction, and the flask (10) is placed in a nuclear meter to measure the marked precipitate.

Description

E HOD OF CONDUCTING RADIOASSAY USING A COMBINED REACTION/FI TER VIAL

Background of the Invention

1. Field of the Invention The present invention relates to radioassay procedures, such as radioimmunoassay, and more particu¬ larly to such procedures in which a radioactively labeled solid phase is derived for measurement.

2. Description of the Prior Art Radioimmunoassay (RIA) procedures have been widely adopted in biological and biochemical analysis in which an assay reaction is conducted in solution to derive a radioactively labeled precipitate or other solid which can be separated from the remaining solu- tion for measurement. For example, one basic RIA method is known as a solid phase double antibody pro¬ cedure and is the same in principle as any double- antibody RIA method. Briefly, in accordance with such method, a reaction is initiated in a vial between (1) a serum sample containing an unknown amount of antigen, (2) a radioactively labeled (1-125) antigen, (3) a primary antibody to the antigen, and (4) a second antibody to the primary antibody. The second antibody is covalently bound to relatively large diameter parti- cles (i.e. 10-20 microns) of microcrystalline cellulose. Upon completion of the reaction, the vial contains some free antigen, both labeled and unlabeled, in a super¬ natant of buffer and serum together with a precipitate complex of the labeled and unlabeled antigen bound to the primary antibody, the primary antibody in turn being bound to the microcrystalline cellulose. Thus, any labeled and unlabeled antigen which has reacted with the primary antibody will be found in the precipi¬ tate, while any labeled or unlabeled antigen which has not reacted with the primary antibody will remain in solution.

OMPI ' Upon completion of the foregoing reaction, the solid precipitate is separated from the supernatant and the radioactive emanations therefrom are counted in a nuclear counting chamber. The number of counts in the precipitate, when expressed as a percent of the total counts of 1-125 antigen added, bears an inverse relationship to the amount of unlabeled antigen ini¬ tially present.

One common approach for separating solid and liquid phases of an assay reaction is centrifugation. By centrifuging the reaction vial, the precipitate is packed in pellet form at the bottom of the vial. The supernatant containing the free antigen is then simply poured off leaving behind the precipitate. The vial containing the precipitate is then placed in a nuclear counting chamber and processed in a conventional manner.

Another approach for separating products of an immunoche ical reaction is filtration. Two filtra¬ tion approaches, referred to generally as external filtration or internal filtration, are illustrated in U.S. Patents 3,825,410 (Bagshawe) and 3,923,463 (Bagshawe et al.). In the external filtration approach, the products of an RIA reaction are drained from the reaction vessel through one of a plurality of filters supported along the length of a conveyor belt. In the internal filtration approach, the reaction vessel comprises at least two stacked sub-units nested together with a filter supported between them. The reaction proceeds in the upper sub-unit. On completion of the reaction, the reactants are forced through the filter either by centrifugation or by connecting a vacuum source to the lower sub-unit. Thereafter, the sub- units of the reaction vessel are disassembled, and the filter is removed and positioned on a conveyor to be transported to a nuclear counter.

While the foregoing methods of conducting

- wipo radioimmunoassay yield satisfactory results, they exhibit a number of serious drawbacks and limitations which reduce their commercial attractiveness. For example, the step of centrifuging in an analysis sequence is not readily automated, and typically requires operator intervention to conduct. Similarly, the internal filtration approach requires operator intervention to disassemble the reaction vessel, remove the filter therefrom, and position the filter on the carrier belt. Moreover, once the filter is separated from its original reaction vessel, it is necessary to institute rigid identifying procedures to ensure that it maintains the identity of the patient whose test was performed in the reaction vessel before disassembly. Similarly, in the external filtration approach where the reaction products are drained from the reaction vessels onto respective filters of a conveyor belt, the same identification procedures are required. A further drawback of the foregoing separation procedures results from the fact that special apparatus, such as centrifuges, filter tapes, conveyors and the like are required which comp¬ licate the task of automating an assay. For the fore¬ going reasons, it would be desirable to derive a method of conducting a radioassay procedure which is compatible with standard methods and apparatus for conducting chemical analysis, which employs a single reaction vial from start to finish of the analysis operation, and which eliminates the need for complicated speciality apparatus for conducting an analysis. Summary of the Invention

The present invention resides in an improved method of conducting radioassay procedures using a combined reaction/filter/ counting vial in a manner which overcomes the drawbacks of prior approaches. The method contemplates the steps of (1) conducting an assay reaction in the combined reaction/filter/counting vial between reactants which include a sample to be assayed and one or more reagents, where at least one of the reactants is radio-labeled, (2) allowing the assay reaction to proceed to derive as one product thereof a radio-labeled solid, (3) withdrawing products of the assay reaction from the vial through the filter medium such that the radio-labeled solid is trapped and re¬ tained in the vial by the filter medium, and (4) intro¬ ducing the vial to radioactive detection means for counting radioactive emanations from the radio-labeled precipitate in the vial. In the preferred embodiment of the invention a plurality of such reaction/filter/ counting vials are supported in a carrier which is readily transported past various operating stations at which the reactants are introduced into the vial and at which the reaction products are withdrawn from the vial through the filter medium. A preferred method for withdrawing material from the vial involves inserting a hollow needle into the vial on the side of the filter medium opposite the assay reaction and connecting a vacuum source to the needle to pull reaction products through the filter and out of the vial through the needle. After thus trapping the radio-labeled preci¬ pitate in the filter medium, the combined reaction/filter/ counting vial is transported to the radioactive detec¬ tion means.

By virtue of the foregoing method, the assay for each sample is conducted in a single reaction/ filter/counting vial from start to finish. The method is readily automated to move a plurality of reaction/ filter/counting vials, supported in one or more vial carriers, the vials along a transport path past appro¬ priate operating stations and is compatible with prior sample handling apparatus for conducting chemical analysis. The vials are transported, operated upon, and introduced into the radioactive detecting means in strict sequence thus maintaining positive sample identity. It is unnecessary to centrifuge in the assay procedure and there is no need to develop specialty apparatus for conveying, handling, identifying and counting filter discs.

Brief Description of the Figures Fig. 1 is a cross-sectional view, taken in a generally vertical plane, through a reaction/filter/ counting vial used in the method of the present invention.

Fig. 2 is a perspective view of a vial carrier for use in the analyzer of Fig. 1.

Fig. 3 is a perspective view of an automated RIA analyzer for practicing the method of the present invention using reaction/ filter/counting vials and vial carriers of Figs 1 and 2.

Fig. 4 is a diagrammatic representation of the generally horizontally disposed vial transport compartment in the analyzer of Fig. 1 and illustrates the direction of movement of vial carriers and the relative location of various operating stations in the analyzer.

Fig. 5 is a partial cross-sectional partial elevational view, taken in a generally vertical plane, illustrating the positioning of a reaction/filter/ counting vial at a filtration station of the analyzer. Fig. 6 is a partial cross-sectional partial elevational view, taken in a generally vertical plane, illustrating the positioning of a reaction/filter/counting vial in the nuclear counting chamber of the analyzer. Description of the Preferred Embodiment

The method of the present invention is designed to use solid phase radioimmunoassay reagent systems such as Digoxin, HTSH, and Cortisol, all as manufac- tured by the assignee of the present invention, but it will be understood that the method may be adapted to other assay and reagent systems. The foregoing specific solid phase systems are described in the following publications: "Solid Phase Digoxin Reagent System for the Radioimmunoassay of Digoxin for In Vitro Diagnostic Use", published by Beckman Instruments, Inc., October 1977; "Solid Phase Cortisol Reagent System for the Radioimmunoassay of Cortisol for In Vitro Diagnostic Use", published by Beckman Instruments, Inc., April 1977; and "Solid Phase HTSH Reagent System for the Radioimmunoassay of Human Thyrotropin for In Vitro Diagnostic Use", published by Beckman Instruments, Inc., September 1977.

Heretofore, the foregoing solid phase RIA tests employed centrifugation to separate bound from free material as previously described. In accordance with the present method, and using a combined reaction/ filter/counting vial 10, as illustrated in Fig. 1, the foregoing procedures may be performed employing a filtration separation step in a manner to be described. The combined reaction/ filter/counting vial 10 comprises an upper tubular vial section 10 the interior volume of which defines an upper or reaction chamber 12 in which a radioassay reaction is to be conducted. The bottom of the vial section 12 is closed by a filter medium, such as a disc of cellulose filter paper, bonded and sealed around its periphery to the bottom annular rim of the vial section in a manner which prevents seepage of fluid around the filter medium. The filter medium

A removable cap 18 is inserted coaxially on the lower end of vial 12 section forming an airtight seal therewith. The interior volume of cap 18 defines a sealed lower or filtration chamber 20 on the side of the filter 16 opposite upper chamber 14. Preferably a plurality of axially extending ribs 22 extend radially inward of lower chamber 20 with the upper edges thereof defining a support surface for a porous mesh disc 24

'A» which abuts filter disc 16 to provide structural support for the filter medium without impeding flow through the filter medium. Cap 18 includes a relatively thin bottom wall 26 closing the lower chamber 20. Bottom wall 26 is adapted to be penetrated during a filtration operation to connect a vacuum source to the lower chamber 20 for drawing fluid from the upper chamber 14 through filter 16 into lower chamber 20 and thence out of the combined reaction/filter/ counting vial 10. The vial components 12, 18 and 24 are con¬ structed of a suitable plastic material, such as poly¬ ethylene or polypropylene, which is inert with respect to the reactants to which it is exposed.

In accordance with an important aspect of the present invention, the combined reaction/filter/counting vial 10 is employed for conducting the aforementioned solid phase radioimmunoassay tests in a manner enabling the radio-labeled solid derived as one product of the assay reaction to be separated from the other reaction products by filter medium 16 and retained within the vial 10. In accordance with a further aspect of the invention, the reaction/filter/counting vial 10 is configured and suitably dimensioned to be inserted into the detecting chamber of a nuclear counter in order to count the radioactivity of the radio-labeled precipitate trapped in the vial. To achieve the foregoing ends, the reactants of a particular assay being run are introduced into the upper chamber 14 of the combined reaction/filter/counting vial and allowed to incubate for a predetermined period of time. The reactants remain in the upper chamber supported by the filter paper and are unable to pass therethrough because of the presence of the air tight seal established at the lower or opposite side of the filter medium by vial cap 18. For the solid phase, double-antibody tests re¬ ferred to above, the reaction develops a complex of the labeled and unlabeled antigen bound to the primary antibody, the primary antibody being in turn bound to the solid particles of microcrystalline cellulose. After the incubation interval, the bottom wall 26 of vial cap 18 is punctured and a source of vacuum connected to lower or filtration chamber 20 to draw the supernatant and other products through the filter medium 16 into lower chamber 20 and out of the vial leaving the solid complex trapped in the vial by the filter medium. The solid complex is trapped by virtue of the fact that the microcellulose particle size is larger than the pore size of the filter medium preventing passage of the solid therethrough. With the labeled solid complex thus separated, the combined reaction/filter/counting vial 10 is inserted into a nuclear counter for measuring the labeled solid complex as previously described. By virtue of the foregoing method, a combined reaction/filter/counting tube 10 is employed for the duration of the assay for the reacting, incubating, filtering, and radioactive counting steps. No centrifugation is necessary during the process nor is there any required disassembly of the combined reaction/ filter/counting vials or transfer of filter medium between various conveyors. The foregoing method may be practiced manually but it readily lends itself to automated implemen¬ tation. Fig. 3 depicts in perspective form an automated RIA analyzer which is adapted for practicing the present method. The analyzer includes a generally horizontal compartment 42 in which a plurality of vial carriers 28 and vials 10 carried thereby are supported and trans¬ ported in a rectilinear path past the various operating stations of the analyzer. In the illustrated analyzer, vial carriers are loaded in compartment 42 at the location designated "in" and are removed from the compartment at the location designated "out". As illustrated in Fig. 2, the vial carriers 28 may be identical in construction to that illustrated in copending U.S. Patent Application Serial No. 754,815, filed December 27, 1976. The vial carrier is of one- piece construction and comprises a generally rectangular base 30 supporting a series of upstanding, generally cylindrical vial holding compartments 32 each of which may receive and support a combined reaction/filter/ counting vial 20. In this regard, each compartment includes an open bottom in the base 30 and an inwardly extending annular rib 34 (Fig. 6) for engaging and sup¬ porting the bottom of a vial 10 in the compartment.

The major components or modules of analyzer 40 include a sample carousel 44, a pipettor-diluter 46, a dispenser 48, and a nozzle assembly 50 which opera- tively coacts with the sample carousel, the pipettor- diluter and the dispenser for introducing sample and required reagents into each combined reaction/filter/ counting vial 10. Nozzle 50 is operatively associated with an assay preparation station 52 where an assay reaction is initiated. Analyzer 40 further includes an incubation station 54, a filtration station 56, and a nuclear counter (gamma) 58. A data reduction system 60 is provided for processing the nuclear counting data, comparing it with data for previously run standards, and outputting meaningful numerical results for each assayed sample.

Fig. 4, in conjunction with Fig. 3, illus¬ trates the path of rectilinear movement of the carrier 28 through the system and designates by appropriate legends the operations performed in the course of analysis. The system is illustrated as including five generally parallel paths A, B, C, D and E, respec¬ tively, for vial carrier movement. Longitudinal move- ment of the carriers in the parallel paths A, B, C, D, and E between ends of compartment 42 and lateral move- ment of each carrier at the end of each path from one path to the other is performed in a conventional manner. Preferably the carriers may be supported for longitudi¬ nal movement in each path on respective pairs of con- veyor belts and may be indexed laterally between the conveyors at the end of each longitudinal path by rotating indexing mechanisms as taught in aforemen¬ tioned copending U.S. Patent Application Serial No. 754,815 filed December 27, 1976. The vial carriers 28 and reaction/filter/ counting vials 10 are loaded into the analyzer at the "in" end of path A and are conveyed by the conveyors to the assay preparation station 52 at the opposite end thereof. At the assay preparation station, nozzle assembly 50 moves laterally over and vertically into a sample cup of carousel 42, picks up a predetermined amount of a serum from the cup, and returns to a posi¬ tion over one combined reaction/filter/counting tube 10 in carrier 28 at preparation station 52. The nozzle assembly is further connected by appropriate conduits and valving (not shown) to pipettor-diluter 46 and dis¬ penser 48 which supply respective appropriate first and second reagents for the designated RIA test. As a result, the nozzle assembly dispenses both a serum sample and the required reagents into a vial 10. The nozzle assembly is then rinsed in a conventional manner, the sample carousel advances to the next serum sample cup, and the next sample is picked up by the nozzle assembly and dispensed with the required reagents into the next vial 10. The process is repeated until all vials 10 in the carrier 28 have received the assayed reactants. At such time the vial carrier is indexed laterally to the beginning of incubation path B and is transported along path B while the reaction proceeds within each vial. At the end of path B the carrier is indexed laterally to the beginning of incubation path C -11- and is then conveyed along path C until reaching the filtration station 56. The time spent in the incuba¬ tion station (paths B and C) is sufficient to allow the reaction in each vial to proceed the required length of time for the particular assay being run.

Fig. 5 illustrates operational details of the filtration station 56. Basically, the filtration station apparatus operates to puncture the bottom wall 26 of a combined reaction/filter/counting vial 10 to connect a vacuum source thereto to draw the assay reaction products through filter medium 16 and out of the vial 10. For this purpose the filtration station includes a hollow needle 62 movable in a vertical direction toward and away from a vial positioned at the filtration station. Needle 62 is supported for vertical movement within a cylindrical shroud 64 which is dis¬ placed vertically by the action of output shaft 66 of a solenoid (not shown). The bore of needle 62 is coupled by a suitable fitting 68 through a conduit 70 connecting the needle to a source of vacuum. The vacuum source and its associated control system may correspond to that illustrated and described in copending U.S. Patent Application Serial No. 883,080 filed March 3, 1978, and assigned to the assignee of the present invention. Shroud 64 is adapted for vertical upward movement in a manner causing the tip of needle 62 to pierce the thin bottom wall 26 of vial 10 thereby entering the lower chamber 20 of the vial but stopping short, of and thus not contacting or penetrating filter medium 16. With the needle tip thus positioned as illustrated in Fig. 5, the vacuum source is actuated to draw reactants from upper chamber 14 through the filter medium into the lower chamber 16 and out through the hollow needle to a suitable waste receptacle. As noted previously, the solid complex is trapped by the filter medium and retained within the combined reaction/filter/

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OMPI counting vial. During application of the vacuum source, a rinse solution probe 72 (Fig. 3) aligned over the top of the vial 10 at the filtration station 56 introduces a suitable rinse solution into the upper chamber. This solution rinses the solid complex before itself being drawn through the filter medium and out through the hollow needle to waste. With the filtration operation thus completed, shroud 64 is retracted downward to remove the needle from the vial, and vial carrier 28 is laterally shifted one position to align the next vial for filtration. When all vials of the carrier have been subjected to the filtration step, the carrier is shifted to the beginning of path D and is conveyed therealong for counting by nuclear 58. The nuclear counter 58 may correspond identi¬ cally to that illustrated and described in the afore¬ mentioned copending application 714,815 also assigned to the assignee of the present invention. After reach¬ ing the end of path D carrier 28 is laterally shifted to position the first vial 10 over an opening 74 in compartment 42. As illustrated in Fig. 6, an elevator mechanism 76, normally positioned below compartment 42, is driven upwardly through opening 74 to engage the bottom of combined reaction/ filter/counting vial 10 and to drive the vial up into the counting chamber 76 of the gamma counter. Radioactive emanations from the solid complex trapped by filter medium 16 are counted in the chamber. After the counting operation, the elevator mechanism is lowered to return the vial to vial carrier 28, and the carrier is indexed laterally one position to align the next vial for counting.

The carrier 28 is then laterally shifted to the beginning of path E and is conveyed toward the "out position" thereof. The order of the reaction/filter/ counting vials 10 in each carrier 28 and the order of the carriers, when more than one is conveyed, is main-

OMP k, ip tained throughout and after the assay.

It will be evident from the foregoing that the present method greatly simplifies and improves the accuracy of radioimmunoassay procedures by conducting the analysis with a single combined reaction/filter/ counting vial 10 from beginning to end and transporting the vial through the necessary operating stations in a manner readily adapted to automated operation without need for operator intervention. The procedure thus eliminates centrifuging completely with its attendant drawbacks of further eliminates any need for vial disassembly and separate processing of filter media. Moreover, while a preferred embodiment of the invention has been illustrated and described, the apparent modi- fications may be made therein without departing from the spirit of the invention as defined in the appended claims.

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Claims

Claims 1. A method of conducting radioassay using a combined reaction/filter/counting vial which includes a filter medium of predetermined pore size comprising the steps of: a. conducting an assay reaction in said vial between reactants which include a sample to be assayed and one or more reagents, at least one of said reactants being radio-labeled; b. allowing said assay ^'reaction to proceed to derive as one product thereof a radio-labeled solid having a particle size exceeding the pore size of said filter medium; c. withdrawing products of said assay reaction from said vial through said filter medium such that said radio-labeled precipitate is trapped and retained in said vial by said filter medium; and d. introducing said vial to radioactive detection means for counting radioactive emanations from said radio-labeled precipitate in said vial.