CA2120481C

CA2120481C - Assay system

Info

Publication number: CA2120481C
Application number: CA002120481A
Authority: CA
Inventors: Steve J. Lackie
Original assignee: Sapidyne Instruments Inc
Current assignee: Sapidyne Instruments Inc
Priority date: 1992-08-03
Filing date: 1993-07-23
Publication date: 2005-12-20
Anticipated expiration: 2013-07-23
Also published as: US5554340A; US6120734A; JP4102837B2; JPH07500191A; EP0606460A1; CA2120481A1; JP2004069716A; JP3534756B2; ATE242996T1; JP2005321416A; EP0606460A4; CA2518356A1; US5372783A; CA2518356C; DE69333050T2; DE69333050D1; DK0606460T3; EP0606460B1; WO1994003104A1; JP2006258824A

Abstract

A system (30) for assaying a fluid sample. The system (30) comprising a lens (33) capable of focussing both excitation and fluorescent radiation, a fluid-flow conducting conduit (42) being provided in the lens (33) extending transversely of the optical axis of and through the focal region of the latter. At least one mechanical screen (38A, 38B) is disposed adjacent the focal region in the conduit (42) to arrest passage of beads (40) as a function of bead diameter. The beads (40) are precoated with at least a moiety of a ligand/conjugated complex and preferably transparent to both the excitation and fluorescent radiation.

Description

2 ~. 2 a 4 ~ 1 PCT/US93/06967 ASSAY SYSTEM
I This invention relates to chemical and biochemical assays, and more particularly to 2 an improved optical apparatus and methods for fluorescent assays.

3 Assays in which aliquots of sample-under-test and one or more reagents are 4 variously reacted in highly specific reactions to form ligand/conjugate complexes such as antigen/antibody or similar complexes which may then be observed in order to assay the 6 sample for a titer of a predetermined moiety from the sample, are well known.
7 Typically, an antibody is used to assay for the presence of an antigen for which the 8 antibody is specific, but such assays have been extended to quantitate haptens such as 9 hormones, alkaloids, steroids, antigens, antibodies, nucleic acids, and fragments thereof, and it is in this broad sense that the term "ligand/conjugate" as used herein should be 11 understood.
12 Sensitive immunoassays typically use tracer techniques in which a tagged 13 constituent of the complex is incorporated, for example in the reagent, the non-complexed 14 tagged reagent then being separated from the complexed reagent. The complexed can be tln'xeaftcr quatttitated by observing a signal from the tag. Radioisotopes, fluorescent and 16 cI>emiluminescent molecules, colorimetric tags, and other markers have been used to label 17 constituents or moieties of the complex, appropriate apparatus being employed to detecx 18 and measure the radiation from the label.
19 In such assays where at least one component of the conjugate complex is initially bound to a solid substrate preparatory to formation of the complex, a basic problem arises 21 because of the typically lengthy time required to bind that component to the substrate.
22 For example, fluorescent assays such as those performed in the usual 96 well microtiter 23 plate, require time in the order of hours for bidding of a component to the solid phase 24 to occur notwithstanding such expedients as heating, shaking and the like.
It will be appreciated that by increasing the surface area of the solid phase made available to 26 binding or coating with a ligand, the binding delay may be considerably reduced.
27 Consequently, the prior art relating to such solid phase assays (such as microtiter well 28 assays; dipstick assays and the like) also teaches using small particles or beads as the 29 solid phase.
Flowing the samph: through a packed particulate bed speeds reactions between ' 31 the sample ligand being assayed and a conjugate immobilized on the surface of the 32 particles. Several factors probably contribute to this enhanced reactivity:
the reduced 33 diffusion distance, the constant stirring of sample due to turbulent flow, and the high 34 density of binding sites in the reaction volume due to the high surface area exposed.

1 Known particle assays include the well-known bead agglutination test including 2 quantitative or semiquantitative slide agglutination and techniques in which the 3 agglutinated beads are separated from non-agglutinated beads by passage through a 4 mechanical filter. Another known particle assay is that described in U.S.
Patent No.
4,780,423 in which particles with controlled porosity having ligand immobilized thereon 6 are incubated in suspension and washed. Washing can involve sedimentation and 7 resuspension of the particles. The resulting fluorescence can be read either from the 8 concentrated or the suspended particles. In yet another known assay, the particles are 9 bound to a membrane or filter through which the sample is then poured. This technique, is believed to have been limited to enzyme~olorimetric detection. Where the particles 11 are incubated in a water suspension, the average diffusion distances which the free ligand 12 in the sample must traverse and the time required to bring complex formation to - 13 completion tee to be quite large.
14 A principal object of the present invention is therefore to provide an improved optical assay system in which the kinetics and sensitivity are improved by increasing the 16 surfacx"area of the solid phase, decreasing diffusion distances, and enhancing the optical 17 coupling among tln; solid phase to the excitation light source and the coupling of the solid 18 phase to the detecxor. Another object of the present invention is to provide a novel flow 19 cell that provides the desired enhancement between the sample and a detector. Yet other objects of the present invention are to provide such an assay system that requires small 21 sample volume and is particularly suitable for assay of whole blood; to provide such an 22 assay system in which the ligand/conjugate reaction is confined within a disposable item 23 that is readily insertable and removable from the optical system of the flow cell; and to 24 provide such an assay system in which all of the components of the desired complex other than the sample moiety to be assayed, are preprovided.
26 Other objects of the present invention will in part be obvious and will in part appear 27 hereinafter. Generally, the foregoing and other objects of the present invention are 28 achieved by a system for assaying a fluid sample; typically employing a tag or label 29 intended to emit electromagnetic radiation when excited, the system comprising a flow cell comprising hollow, light-transparent conduit means adapted for fluid flow 31 therethrough, and one or more separate porous masses of light-transparent material 32 disposed in the conduit means, the porosity of the mass of transparent material being 33 selected to permit fluid flow of the sample therethrough, at least a moiety of a respective 34 ligand/conjugate complex e.g. a specific-binding ligand, being immobilized, as by precoating, on the surfaces of each mass. .

r , 1 In one embodiment, the mass comprises a plurality of particles preferably 2 substantially transparent to light, particularly, where the complex formed includes a 3 fluorescent label, transparent to both radiation required to excite fluorescent and the 4 excited fluorescent. The particles are typically beads dimensioned within a specified S range of diameters and can be preformed, as by sintering or the like.
Alternatively, the 6 mass can be formed by accretion against a fluid-porous barrier means disposed in the 7 conduit means. In the latter case, the barrier means is disposed within the conduit means 8 so as to define at least one wall of a chamber, the porosity of the barrier means being 9 sufficiently smaller than said range so that particles entrained in a fluid flow through the conduit means are trapped by the barrier means and accrete to form the porous mass in the 11 chamber.
12 A preferred embodiment of the present invention includes focussing optical lens 13 means through which the conduit means forms a hollow, tubular passage extending 14 transversely to the optical axis of and through the focal region of the lens means.
Typically, the lens means comprises a plurality of lenses and the conduit means extends 16 though one of those lenses. Where the system is to be used with a tag or label intended to 17 emit electromagnetic radiation when excited, the lens means must be capable of focussing 18 both the excitation and the emission radiation.
19 According to one aspect of the present invention, there is provided an apparatus comprising, in combination: a light-transparent conduit means for allowing fluid flow of a 21 fluid sample therethrough; and a porous mass of light-transparent material disposed in said 22 conduit means, the porosity of said mass being selected to permit fluid flow of said fluid 23 sample therethrough, said mass having immobilized thereon at least a moiety of a 24 ligand/conjugate complex, and characterized by: measuring means positioned relative to said porous mass and arranged to quantitatively measure an amount of electromagnetic 26 radiation emanating therefrom.
27 According to another aspect of the present invention, there is provided a method of 28 assaying a fluid sample by measuring radiation emitted from a ligand/conjugate complex, 29 said method comprising the steps of: providing an apparatus as described herein; treating said porous mass, including flowing at least said fluid sample therethrough, so as to create 31 said ligand/conjugate complex on surfaces of said particles; stimulating said complex so 3a 1 that characteristic radiation arises therefrom; focusing said characteristic radiation by 2 focusing optical lens means having a focal region; and measuring the amount of said 3 characteristic radiation that is emitted.
4 The invention accordingly comprises the apparatus possessing the construction, combination of elements and arrangement of parts, and the method comprising the several 6 steps and the relation of one or more of such steps with respect to each of the others, all as 7 exemplified in the following detailed disclosure, and the scope of the application of which 8 will be indicated in the claims.
9 For a fuller understanding of the nature and objects of the present invention, reference should be had to the following detailed description taken in connection with the 11 accompanying drawings in which like numerals in the several drawings are employed to 12 denote like parts, and wherein:
13 Fig. 1 is a diagrammatic representation, in cross-section, of assay apparatus 14 embodying the principles of the present invention;
1 S Fig. 2 is a schematic cross-section of one embodiment of the flow cell of the 16 present invention;
17 Fig. 3 is a transverse cross-section of the flow cell of Fig. 2;
18 Fig. 4 is a schematic cross-section of a variation of the flow cell of Fig.
2;
19 Fig. 5 is a schematic cross-section of another variation of the flow cell of the present invention;

WO 94/03104 . PCT/US93/06967 ~~.~~~$1 1 Fig. 6 is a schematic cross-section of a variation of the flow cell of Fig.

5;
2 Fig. 7 is a schematic cross-section of another variation of the flow cell of Fig.
3 5; and 4 Fig. 8 is a schematic cross-section of yet another embodiment of the flow cell of the present invention.

6 In Fig. 1 there is shown exemplary apparatus 20 far assaying a fluid sample and 7 which may typically employ an optical system including light source 22 for providing 8 excitation radiation, light detector 24 for detecting light stimulated by the excitation 9 radiation, beam spliaer means such as dichroic or semitransparent mirror 26 and collimator means 28. The embodiment of Figs. 1, 2 and 3 will be described, for ease 11 of exposition, for use particularly in the context of fluorescence immunoassay, but it 12 should be understood is not so limited. The term "light" as used herein will be 13 understood to include wavelengths in the visible spearum as well as those in the hear 14 infra-red and ultraviolet as well. Similarly, the term "excitation" will be understood to itxlude excitation of fluorescence, polarized or not, as by radiation, excitation of 16 chemiluminescxnce by c>temicat agents; emission by reflection of light from chromogens, 17 and the like.
18 The foregoing elements of the optical system are typically disposed in a frame 19 (not shown) in fixed optical Telationship to one another, as described more fully hereinafter. The invention further includes a flow cell 30, shown particularly in enlarged 21 form in Figs. 2 and 3, and in this embodiment, formed from a focussing optical lens 22 means 32 shown as a compound lens system including solid focussing lens 33, typically 23 made of glass, high molecular weight polymer or the Like. Lens 33 is characterized by 24 having an elongated hollow channel or fluid-flow conducting conduit 34 therein directed transversely to the optical axis of lens 32 and comprising a tubular passage, typically of 26 circular cross-section, through lens 33. At least a portion of such cylindrical conduit, 27 reaction chamber 36, is disposed at the focal region of lens means 32.
28 Thus, for example assume that fluid containing a ligand that can be excited, per 29 se or through an appropriate tag, into emission such as fluorescence, traverses chamber 34 and is appropriately excited into emission there by excitation radiation focussed onto 31 chamber 34 by lens means 32. That fluorescent emission is then directed by lens means 32 to detector 24 where, assuming that the detector for example is electrical, appropriate 33 electrical signals are produced and can be assessed to evaluate the fluorescence.
34 In order to provide a better signal-to-ligand ratio, the embodiment shown in Fig.
4 includes mechanical, fluid-porous barrier or screen 38 dimensioned and disposed in WO 94/03104 2 ~ z a ~ s 1 PCT/US93/06967 1 conduit 34 adjacent the focal region of lens means 32 so as to arrest transport of particles 2 or beads 40 of predetermined size in a flow stream through the conduit. Such beads are 3 substantially transparent to both the excitation radiation and the excited fluorescence, and 4 to that end are typically formed of polymethylmethacrylate, styrene-divinyibenzene S copolymer or the like. Beads 40 are coated with at least a moiety of the antibody/antigen 6 complex, e.g. a specific-binding ligand, for example an antigen and an antibody thereto, 7 disposed at least on a portion of the surface of the bead.
8 The mesh or porosity of screen 38 is selected to allow free flow of sample fluid 9 and its constituents therethrough while arresting flow of the coated beads, and thereby accreting a mass of beads 40 against the screen and in the focal region of the lens means 11 32. The particle size of the beads is selecxed to be minimized, provided however that 12 when a mass of beads is accreted against screen 38, the sample constituents may still pass -13 freely through the accretion mass. Typically, a bead size that works well with whole 14 blood as a sample is in the range'of 50 hem to 250 Vim, preferably around 98 /cm. Bead size, of course, depends to some extent on the nature of the sample (e.g.
blood, food, 16 urir~, process stream and the like). Mesh size, of course, depends upon the range of 17 diameters of tl~ beads to be employed in the system, but typically, for beads of about 98 18 hem diameter, a mesh size of about 50 ~,m is appropriate. Thus, as sample fluid is flowed 19 through conduit 34, it must pays through the interstices of the accreted mass of coated beads 40, resulting in a very small diffusion distance over which the assayed moiety must 21 pass to complex with the coating on the beads. This small diffusion distance, coupled 22 with the long, tortuous path of the sample through the accreted mass and the high surface 23 to volume ratio of the beads, enables very efficient scavenging of the assayed moiety 24 from the sample. This characteristic of the present invention is significant inasmuch as the diffusion time is reduced by the square of the diffusion distance. It should also be 26 noted that the entire solid phase is contained in the accreted mass, a very small volume 27 (e.g. about 0.02cm' for a typical conduit of 0.18 cm diameter), and is "immersed" in lens 28 32 thus providing a high numerical aperture, optical coupling between the excitation and 29 detection systems. Because the fluorescent signal is increased by the fourth power of the numerical aperture, high numerical aperture optical coupling is very important.
31 In operation of the invention shown in Fig. 4, a quantity of beads 40 are preferably 32 preloaded with an appropriate ligand immobilized onto the bead surfaces by adsorption 33 or other known immobilizing techniques and suspended in a suspending fluid.
Where the ' 34 beads will ordinarily not readily form a stable suspension in the suspending fluid, they 3~ may be placed into a vortexer (not shown') or similar mixer which maintains the beads WO 94/03104 ~ ~ ~ R PCT/US93/06967 1 in a suspension, typically aqueous, by agitation. A desired portion of the bead suspension 2 is sucked out of the vortexer as by a pump (not shown) and injected into conduit 34 3 where the flow of the beads is arrested by screen 38, creating an accretion or mass of 4 beads 40 within reaction chamber 36. An aliquot of sample solution being assayed is S then flowed through conduit 34 and the mass of beads 40 in reaction chamber 36, 6 effecting the formation of a ligand/conjugate complex on the surface of the beads. As 7 is well known, for competitive assays, prior to flowing the sample solution through the 8 flow cell, typically the sample solution is first treated with a tagging reagent and allowed 9 to inarbate. Where the assay is a sandwich assay, the sample solution is passed through the flow cell, then tagged antibody is passed through the cell, and the bead mass is I1 subjecxed to a wash step. As is well known in the art, a tagged, typically fluorescent, 12 component may be either the complement or conjugate to or an analog of the immobilized -13 ligand, depending upon whether a competitive or sandwich assay is to be performed. The 14 tag or label is typically a fluorescent dye such as a fluorescein dye, acridine dye or the like, all as well known in the art. In either case, the resulting ligandlconjugate complex 16 should include desired dye moi~ies bound to the complex. Flowing a wash buffer 17 through the bead mass then washes out any unreacxed materials and particularly any free 18 dye components, leaving only those dyed moieties as are immobilized on the beads.
19 Light source 22 is then acxivatEd to generate excitation light beam 23 (shown in broken lines) which, in turn, directed to mirror 26 by collimating lens 28 so that the collimated 21 beam is reflected onto lens means 32. The latter focusses the excitation beam to a focal 22 region at which the mass of beads 40 in reaction chamber.36 is located, and the excitation 23 radiation excites the fluorophores on beads 40 into fluorescence. That fluorescence is 24 transmitted through lens 32 and directed through beam sputter mirror 26 to detector 24.
After measurements are made, the mass of beads 40 can be readily removed from 26 reaction chamber 36 simply by back-flushing through conduit 34.
27 As thus described, the technique of filling the reaction chamber from a suspension 28 or pool of preloaded beads is clearly amenable to automation, where the components for 29 specific assays, such as the type of preloaded beads, sample solution, tagging reagent and the like, are selectable by appropriate valves controlling the flow of materials from 31 respective storage containers. However, the present invention also is readily adaptable 32 for more portable systems in which the bead mass and reagents are disposables.
33 For example, while conduit 34 is shown in Fig. 3 to be simply a passageway 34 through the focal region of lens means 32 transverse to the optical axis of the lens, in the 35. embodiment shown in Fib. ~, conduit 34 is formed of elongated bore 34A of uniform 1 diameter provided similarly through lens 33 and elongated light-transparent tube 42 2 having a uniform diameter slightly less than that of bore 34A so that tube 42 may be 3 inserted and removed from the bore. Screen 38 is so disposed within tube 42 that the 4 latter can be positioned within bore 34A adjacent the focal region of the lens.
In yet another embodiment of the flow cell of the present invention, as shown in 6 Fig. 6, conduit 34 is similarly formed of elongated bore 34A of uniform diameter 7 through lens 33 and elongated light-transparent tube 42 having a uniform diameter 8 slightly less than that of bore 34A so that tube 42 may be inserted and removed from 9 the bore. Screens 38A and 38B are so disposed within tube 42 in spaced-apart relation to to one another so as to define reaction chamber 44 within the tube. As in the 11 embodiment of Fig. 5, reaction chamber 44 can be positioned within bore 34A
adjacent 12 the focal region of the lens. Included within chamber 44 is a plurality of beads 40 13 dimensioned within a specified range of diameters, the mesh of screens being 14 sufficiently smaller than the range of bead diameters so that the latter are trapped by the screens in chamber 44 to form a porous mass of positionable substantially at the lens 16 focal region. The beads in the embodiment of Fig. 6 are preferably precoated with the 17 desired specific binding ligand before installation in chamber 44.
18 In both the embodiments of Figs. 5 and 6, it will be appreciated that tubes 42 are 19 preferably readily insertable and removable in and from bore 34 or 34A as the case may be, hence may be considered to be "disposables". Particularly, the "disposable" shown 21 in Fig. 6 lends itself to laboratory preloading and packaging in hermatically sealed 22 containers from convenient distribution and use. In both the emobidments of Figs. 5 23 and 6, the materials forming both lens 32 and tube 42A are selected so that the 24 respective indices of refraction thereof are substantially matched. In order to provide the optimum optical coupling between tube 42A and lens 32, a refractive index-26 matching fluid is preferably disposed around the tube in the interspace between the tube 27 and the interior wall of bore 34A.
28 It should be understood that bead mass 40 of the embodiment of Fig. 6 can be 29 formed by, for example, the same technique used to create the bead mass of Fig. 5, i.e.
by flowing a suspension of beads through conduit 42 to accrete against a screen such as 31 38B, the other screen then being emplaced to capture the bead mass.
Alternatively, the 7a 1 porous bead mass may also be formed of a plurality of bead adhered lightly to one 2 another as by sintering or adhesives. For example, the bead mass can be formed by 3 providing a thick layer of beads which may be free-standing, or by coating a porous 4 substrate or forming a sandwich between a pair of porous substrates, with the thick layer ~~.~a~~l:
_g_ 1 of beads, which bead layers include a minor amount of adhesive that wilt not materially 2 reduce the porosity of the resulting mass. After curing, the coating can be precoated with 3 an appropriate specifically reactive ligand and minute cylinder of the coating punched out 4 and inserted into appropriately dimensioned tubes 42. Alternatively, sheets of high-s molecular weight polymeric material of the desired porosity are commercially available, 6 and after treatment to immobilize the requisite ligand within the porous structure, can be ' 7 punched to produce the desired cylinders for insertion into tubes 42. Thus, one may 8 provide a plurality of bead masses, each coated with a different ligand. The resulting 9 plurality of bead masses can be emplaced in a single tube 42, as shown in Fig. 7, so that one may assay a sample flowing through the tube for several different ligands separately 11 but substabtially simultaneously.
12 As shown in Fig. 8, conduit 34 can be formed in part as a shallow elongated _ 13 channel 34B or hemi-tubular portion of, for e~tample, semicircular cross-section cut or 14 molded into planar surface 48 of lens 33 which extends perpendicularly to the optical axis of the lens and through the focal region of lens means 32. The remainder of conduit 34 16 is formed by another l~mi-tubular elongated channel 34C, similarly of semiciratlar cross-17 secxion, provided in plats 50. The laser is attached to lens 32 adjacent surface 48, 18 typically by hinging 52 such that plate 50 can be rotated to match channels 34C and 34B
19 into coaxial relation to form a'combined conduit of substantially circular cross-section.
In the preferred embodiment, the inner surface of channel 34C is provided with highly 21 reflexive coating 54.
22 Since certain changes may be made in the above process and apparatus without 23 departing from the scope of the invention herein involved, it is intended that all maser 24 contained in the above description or shown in the accompanying drawing shall be interpreted in an illustrative and not in a limiting sense.

Claims

WE CLAIM:

1. An apparatus comprising, in combination:
a light-transparent conduit means for allowing fluid flow of a fluid sample therethrough; and a porous mass of light-transparent material disposed in said conduit means, the porosity of said mass being selected to permit fluid flow of said fluid sample therethrough, said mass having immobilized thereon at least a moiety of a ligand/conjugate complex, and characterized by:
measuring means positioned relative to said porous mass and arranged to quantitatively measure an amount of electromagnetic radiation emanating therefrom.

2. The apparatus as defined in claim 1 wherein said porous mass comprises a plurality of particles dimensioned within a specified range of diameters; said apparatus further including:
fluid-porous barrier means disposed within said conduit means, the porosity and location of said barrier means being selected so that said particles are trapped by said barrier means to form said porous mass.

3. The apparatus as defined in claim 2 wherein:
(a) said barrier means comprises at least a pair of screens spaced apart from one another so as to define a reaction chamber within said conduit means, the mesh of said screens being sufficiently smaller than said range of diameters so that said particles are trapped between said screens in said chamber to form said porous mass, said particles having immobilized on the surfaces thereof at least a moiety of a ligand/conjugate complex; or (b) in combination:

said barrier means comprises a plurality of pairs of screens, the screens of each said pair being spaced apart from one another so as to define a reaction chamber within said conduit means, so that said plurality of pairs defines a plurality of reaction chambers within said conduit means, each said reaction chamber including a plurality of particles dimensioned within a specified range of diameters, the mesh of said screens being sufficiently smaller than said range of diameters so that said particles are trapped by said screens in each said reaction chamber to form a porous mass in each reaction chamber, the particles of each said porous mass having immobilized on the surfaces thereof at least a moiety of a ligand/conjugate complex.

4. The apparatus as defined in claim 1, wherein each moiety of a ligand/conjugate complex comprises a plurality of distinct segments, each segment being disposed in a different region of said conduit means.

5. The apparatus as defined in any one of claims 1 to 4 and including:
a focusing optical lens means; and the conduit means being of uniform cross-sectional dimension disposed within said lens means and extending transversely to an optical axis of said lens means through a focal region of said lens means focusing said light rays by refraction and said lens means being positioned to direct rays that emanate from within said conduit means to said measuring means.

6. The apparatus as defined in claim 5 wherein said lens means comprises a plurality of lenses and said conduit means comprises a cylindrical passage extending through one of said lenses.

7. The apparatus as defined in claim 5 including a mechanical, fluid-porous barrier means dimensioned and disposed adjacent to said focal region so as to block flow of particulates of or above a predetermined size through said conduit means.

8. The apparatus as defined in claim 5 including a first elongated hemi-tubular channel in said lens means and a second elongated hemi-tubular channel, said channels being mateable with one another to define said conduit means.

9. The apparatus as defined in claim 8 wherein said second hemi-tubular channel has a light reflective surface on the concave portion thereof; and wherein one edge of said first channel is hingedly connected to an edge of said second channel.

10. The apparatus as defined in claim 7 wherein said conduit means comprises a cylindrical passage having a light-transparent tube positioned therein, said barrier means being disposed within said tube; and wherein (a) said barrier means comprises a pair of screens spaced apart from one another so as to define a reaction chamber within said tube; and including a plurality of particles dimensioned within a specified range of diameters, the mesh of said screens being sufficiently smaller than said range of diameters so that said particles are trapped by said screens in said reaction chamber to form a porous mass positionable substantially at said focal region; or (b) said lens means and said tube have indices of refraction that are substantially matched, and the apparatus includes a refractive index-matching fluid disposed around said tube and between said tube and said passage.

11. The apparatus as defined in claim 6 wherein one portion of said passage is formed as an elongated hemi-tubular first channel in said lens means and the remaining portion of said passage is formed as an elongated hemi-tubular second channel, matched to said first channel.

12. The apparatus as defined in claim 7 including a plurality of particles dimensioned within a specified range of diameters, said fluid-porous barrier means having pores of lesser diameter than said range of diameters so that said particles are accreted in said conduit means against the fluid porous barrier means to form a porous mass disposed substantially at said focal region.

13. The apparatus as defined in claim 5 and suitable for assaying a fluid sample including an excited tag that emits electromagnetic radiation when flowed through said conduit means;
the apparatus further comprising a fluid-porous barrier means disposed adjacent to said focal region in said conduit means for limited passage of particles through said fluid-porous barrier means as a function of particle size.

14. The apparatus as defined in claim 1 or 13 including means for exciting emission of said electromagnetic radiation.

15. The apparatus as defined in claim 14 wherein said means for exciting said emission comprises a source of excitation radiation and further comprising means for directing said excitation radiation at said conduit means at said focal region.

16. The apparatus as defined in claim 15 wherein said emission is fluorescent radiation.

17. The apparatus as defined in claim 15 including a plurality of particles dimensioned within a specified range of diameters, said fluid-porous barrier means having pores of lesser diameter than said range of diameters so that said particles are accreted in said conduit means against said fluid-porous barrier means to form a porous mass disposed substantially at said focal region.

18. The apparatus as defined in claim 17 wherein said particles are substantially transparent to both said excitation radiation and said fluorescent radiation, and are at least partly coated with immobilized specific binding ligand.

19. The apparatus as claimed in claim 18 in which the ligand has formed a complex in a ligand/conjugate reaction, said complex being tagged with molecules that fluoresce when excited by appropriate excitation radiation.

20. Method of assaying a fluid sample by measuring radiation emitted from a ligand/conjugate complex, said method comprising the steps of:
providing the apparatus of claim 2;
treating said porous mass, including flowing at least said fluid sample therethrough, so as to create said ligand/conjugate complex on surfaces of said particles;
stimulating said complex so that characteristic radiation arises therefrom;
focusing said characteristic radiation by focusing optical lens means having a focal region; and measuring the amount of said characteristic radiation that is emitted.

21. The method of claim 20 wherein (a) said treating includes coating said particles at least on a portion of the surface thereof with at least a moiety of said complex;
tagging said complex with a fluorescent label; and flowing through said conduit means and said porous mass a wash fluid to separate unreacted conjugate and fluorescent label from the tagged complex; or (b) said stimulating includes directing excitation radiation onto said tagged complex; and said characteristic radiation comprises fluorescence arising from the excited tagged complex and passing through said focusing optical lens means.

22. The method of claim 20 whereby said light-transparent conduit means consists of a tube and said fluid-porous barrier means comprises a pair of screens spaced such that said screens define a reaction chamber within said tube containing said porous mass of said plurality of particles; and wherein said tube is inserted into a cylindrical passage transversely through said lens means so that said reaction chamber lies within said focal region.

23. The apparatus of claim 1, wherein said electromagnetic radiation is emitted from a radioisotope.

24. The apparatus of claim 1, wherein said electromagnetic radiation comprises fluorescent radiation.

25. The apparatus of claim 1, wherein said electromagnetic radiation comprises chemiluminescent radiation.

26. The apparatus of claim 1, wherein said measuring means comprises a fluorimeter.

27. The apparatus of claim 1, wherein said measuring means comprises a photomultiplier tube.