CA2063986A1 - Method and apparatus for rapid immunoassays - Google Patents
Method and apparatus for rapid immunoassaysInfo
- Publication number
- CA2063986A1 CA2063986A1 CA 2063986 CA2063986A CA2063986A1 CA 2063986 A1 CA2063986 A1 CA 2063986A1 CA 2063986 CA2063986 CA 2063986 CA 2063986 A CA2063986 A CA 2063986A CA 2063986 A1 CA2063986 A1 CA 2063986A1
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- CA
- Canada
- Prior art keywords
- holes
- plate
- chamber
- membrane
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
- B01L3/50255—Multi-well filtration
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5302—Apparatus specially adapted for immunological test procedures
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Clinical Laboratory Science (AREA)
- Automatic Analysis And Handling Materials Therefor (AREA)
Abstract
An immunoassay apparatus is disclosed for the quantitative determination of the concentration of a target ligand in a liquid sample. The apparatus contains top (10) and middle (14) plates having holes (12, 18). The holes in the two plates are in axial alignment and the holes in the middle plate have sidewalls (22) that extend below the bottom surface of the middle plate. A liquid permeable membrane (16) is placed between the plates. The apparatus contains a bottom chamber (40) open to the bottom surface of said middle plate, a vacuum port (42) and can hold a microtiter plate containing a plurality of wells (52). Liquid is placed in the holes in the top plate and vacuum is created in the chamber. The liquid is drawn at a controlled rate directly through the membrane without lateral dispersion, through the holes in the middle plate and, into the bottom chamber.
Description
WO9l/02073 "j~, PC~/~S90/040~6 r~ f l r "Immunoassa~ ~'acuum Chamber With ~icrotiter Plate"
Field of Invention The present invention relates to immunoassay procedures and, in particular, to quantitative immunoassay procedures which can be rapidly accomplished in su~stantially less tlme than heretofore obtainable.
BacXqround Immunoassay procedures have for many years : provided sensitive diagnostic tools for the detection of a variéty of substances, generally referred to as ligands. Such procedures are described in a number of : articles and textsj an example of which is Reviews on : Immunoassay Technoloqy, Ed. S. B. Pal, Pub. Chapman &
15 Hall, 1988.
~ One type of immunoassay procedure, commonly referred to as E~ISA~ utilizes a solid support such as the well in a plastic plate in accomplishing the assay. A receptor for a target ligand is bound to the : 20 ~ solid Cupport. A liquid sample containing the liyand, having speci~icity for the bound receptor, is then applied to the plate. Following washing and incubation procedures, an enzyme conjugate having binding specificity for the ligand is added to the :
:
.
PCI`/~lS90/()qO86 well. After further rinsing, ~ substrat~ is added which develops color on contact with the bound enzyme, the amount of color developed ~eing dependent upon the amount of bound con~ugate present which, in turn, i5 indicative of the àmount of target ligand bound to the support. Thus, by measuring the amount of color development and correlaking such against known standards, the concentration of ligand in the sample can be determined. In general, conventional ELISA
procedures take on the ~3rder of five hours or more.
Recently, it has been suggested that the ELISA
procedure can be accelerated by utilizing immunofiltration (Ijsselmuiden et al., Journal of Immunolo~ical Method, 119 (1~89) 35-43 and Eur. ~.
Microbiol. 6, (1987) 281). In the disclosed procedurej a nitrocellulose filter is pre-coated with an antigen. Thereafter, a solution containing the target ligand, in this case an antibody, is drawn through the filter followed by rinsing solutions.
Finally, either an enzyme-labeled antibody (Ijsselmuiden (1987)) or 125I-labeled protein A
(Ijsselmuiden (1989)), both of which have binding specificity for the target ligand, is applied and drawn through the filter to detect the target antibody bound to the antigen on the nitrocellulose filter.
The device used to accomplish the above described assay is illustrated in the 1989 Ijsselmuiden artic1e.
It consists of three blocks of perspex which are clamped together during the assay. The bottom section has an external outlet and a valve, and constitutes a reservoir attached to the upper sections. The middle and top sections, designed to accommodate the nitrocellulose filter between them, contain 32 corresponding holes with a diameter o~ 5 mm and neoprene 1'0ll rings facing the nitrocellulose sheet to prevent lateral flow.
WO~l/02073 PCr/US90/0~0~6
Field of Invention The present invention relates to immunoassay procedures and, in particular, to quantitative immunoassay procedures which can be rapidly accomplished in su~stantially less tlme than heretofore obtainable.
BacXqround Immunoassay procedures have for many years : provided sensitive diagnostic tools for the detection of a variéty of substances, generally referred to as ligands. Such procedures are described in a number of : articles and textsj an example of which is Reviews on : Immunoassay Technoloqy, Ed. S. B. Pal, Pub. Chapman &
15 Hall, 1988.
~ One type of immunoassay procedure, commonly referred to as E~ISA~ utilizes a solid support such as the well in a plastic plate in accomplishing the assay. A receptor for a target ligand is bound to the : 20 ~ solid Cupport. A liquid sample containing the liyand, having speci~icity for the bound receptor, is then applied to the plate. Following washing and incubation procedures, an enzyme conjugate having binding specificity for the ligand is added to the :
:
.
PCI`/~lS90/()qO86 well. After further rinsing, ~ substrat~ is added which develops color on contact with the bound enzyme, the amount of color developed ~eing dependent upon the amount of bound con~ugate present which, in turn, i5 indicative of the àmount of target ligand bound to the support. Thus, by measuring the amount of color development and correlaking such against known standards, the concentration of ligand in the sample can be determined. In general, conventional ELISA
procedures take on the ~3rder of five hours or more.
Recently, it has been suggested that the ELISA
procedure can be accelerated by utilizing immunofiltration (Ijsselmuiden et al., Journal of Immunolo~ical Method, 119 (1~89) 35-43 and Eur. ~.
Microbiol. 6, (1987) 281). In the disclosed procedurej a nitrocellulose filter is pre-coated with an antigen. Thereafter, a solution containing the target ligand, in this case an antibody, is drawn through the filter followed by rinsing solutions.
Finally, either an enzyme-labeled antibody (Ijsselmuiden (1987)) or 125I-labeled protein A
(Ijsselmuiden (1989)), both of which have binding specificity for the target ligand, is applied and drawn through the filter to detect the target antibody bound to the antigen on the nitrocellulose filter.
The device used to accomplish the above described assay is illustrated in the 1989 Ijsselmuiden artic1e.
It consists of three blocks of perspex which are clamped together during the assay. The bottom section has an external outlet and a valve, and constitutes a reservoir attached to the upper sections. The middle and top sections, designed to accommodate the nitrocellulose filter between them, contain 32 corresponding holes with a diameter o~ 5 mm and neoprene 1'0ll rings facing the nitrocellulose sheet to prevent lateral flow.
WO~l/02073 PCr/US90/0~0~6
2 o~ 9,8 6 While Ijsselmuiden appears to describe an immunoassay procedure which has the a~vantage of rapidity over prior procedures, only the procedure utilizing 125I detection is quantitative. The enzyme-labeled antibody system (Ijsselmuiden (1987)) yieldsonly a qualitative determination o~ target ligand. A
quantitative procedure having the advantages of rapidity and not necessitat:ing the use of radioactively labeled detection reagents such as 1~5I
would be desirable. In addition, a procedure having the foregoing attributes which also can utilize commercially available microtiter plates and associated readers for determination of color development would be advantageous.
Summary of the Present Invention In accordance with the present invention, there is provided a procedure and related apparatus for rapidly and quantitatively accomplishing immunoassays without the necessity for using a radioactive detection system. Further, in accordance with this - invention, standard microtiter plates and associated readers can be used to quantitate the results of the assay.
The present invention embodies the features of filtration, such as illustrated by Ijsselmuiden. But, in addition thereto, it provides means for collecting the colored reaction product o~ enzyme and substrate in ~ fashion that permits quantitative measurement of color development in a microtiter plate system to achieve quantitation of target ligand~
Accordingly, the present invention provides an apparatus which can be used for colorimetric ligand-receptor assay procedures to quantitatively determine the concentration of a target ligand in a liquid .. ,. , . . , . , , . . , i
quantitative procedure having the advantages of rapidity and not necessitat:ing the use of radioactively labeled detection reagents such as 1~5I
would be desirable. In addition, a procedure having the foregoing attributes which also can utilize commercially available microtiter plates and associated readers for determination of color development would be advantageous.
Summary of the Present Invention In accordance with the present invention, there is provided a procedure and related apparatus for rapidly and quantitatively accomplishing immunoassays without the necessity for using a radioactive detection system. Further, in accordance with this - invention, standard microtiter plates and associated readers can be used to quantitate the results of the assay.
The present invention embodies the features of filtration, such as illustrated by Ijsselmuiden. But, in addition thereto, it provides means for collecting the colored reaction product o~ enzyme and substrate in ~ fashion that permits quantitative measurement of color development in a microtiter plate system to achieve quantitation of target ligand~
Accordingly, the present invention provides an apparatus which can be used for colorimetric ligand-receptor assay procedures to quantitatively determine the concentration of a target ligand in a liquid .. ,. , . . , . , , . . , i
3 l'Cr/US~0/04086
4 . ' sample. sriefly described, the apparatus contains ~
top member, a middle member and a bottom member in a sandwiched relatl`onship. The top and middle members are plates having holes therethrough and between which a membrane can be placed. When the plates are placed one on top of another the holes are in axial alignment and the cross-sectional area o~ the holes at the hottom surface of the top plate is greater than the cross-sectional area of the holes at the top surface of the middle plate. ~urthermore, the sidewalls of the holes in the middle plate extend below the surface of the plate. As described later, the holes in the middle plate are preferably contained within tubes, herein termed cannulas, which are inserted through openings initially formed in the middle plate.
The bottom member of the apparatus is a collection chamber which has an opening on the upper side which faces the bottom surface of the middle plateO The chamber contains a port through one of its surfaces so that a YacUUm can be created within the chamber. The chamber contains means for accepting a microtiter plate containing a plurality of wells. In the assembled apparatus, the ends of the sidewalls of the holes which extend beneath the bottom surface of the middle plate are located within the wells of the microtiter plate. The apparatus contains means ~or securing the three members together in a vacuum type relationship when a membrane is positioned between the top and middle plates.
In use, the apparatus described above is first assembled with a liquid permeable membrane, to which a receptor can be bound, placed between the top and middle plates, and without the plate containing the wells being positioned in the bottom chamber. The membrane can either have the receptor already bound thereto or a solution containing the receptor can be . . . , : .
WO9l/0~073 PCr/US~0/0~086 ¦
9;8 6 added to the holes in the top plate. In the later instance, vacuum is then created in the chamber with, for example, a peristaltic pump, so that the solution can be pulled past the membrane at a highly controlled consistent rate.
Having the receptor now bound to the membrane, the liquid sample containiny the target ligand is added to the holes in the top plate. As above described, vacuum is applied and the liquid is drawn directly through the membrane, through the holes in the middle plate, and then discharged into the collection chamber. As the liquid sample passes through the membrane, the ligand is bound to the receptor on the membrane. Thereafter, a solution containing an enzyme conjugate which has binding specificity for the target ligand is drawn through the membrane and is, in turn, bound to the target ligand.
This step is then followed by a washing step to remove enzyme conjugate which did not bind to the ligand.
After the above described steps, the bottom chamber is separated from the top and middle plates, which remain secured together, and is emptied of any liquid which may be present from previous steps.
Then, the microtiter plate is positioned in the bottom chamber and the apparatus is reassembled with the sidewall extensions o~ the middle plate now extending into the wells of the microtiter plate.
A solution contaihing a substrate for the bound enzyme, which on reaction therewith gives a colored product, is then add~d ~o the holes in the top plate and drawn through the membrane, through the holes in the middle plate and, in turn, is deposited into the wells of the microtiter plate. Sufficient substrate solution is uti.lized so that on collection in the wells the solution extends above the bottom ends of the sidewall extensions. This feature is important '; ~'', ~ . , , ,, ,,,'j ~,' '. . ' ' ', I ' '' ' ' ~ .::' .: -' .
' ': ' , , ' , '. ' ' . ' .. .
., ' . ' .... ' ' ,' '' ": ' ' . ' ' , , WO~1/02073 l'CT/US9~/04086 ~; , ,390 in order to insure that the volume of solution transferred from the holes in the upper plate to the wells in the micr~ iter plate is the same for each set of holes and ~ells in the apparatus. In turn, this permits measurement of well to well differences in color intensity which correlate to differences in bound ligand. After collection is complete, the bottom chamber is again separated from the remaining structure, the microtiter plate removed from the chamber and the development of color of the solution in the wells determined.
Brief Description of the Drawln~s FIG. 1 is an exploded perspective view showing the apparatus of the present invention with certain parts omitted and with a membrane included.
FIG. Z is an exploded side elevation with certain parts broken away and shown in section.
FIG. 3 is a cross-section taken vertically through the apparatus.
FIG. 4 is an enlargement, partly in section, of one of the clamps.
FIG. 5 is a top plan view of the assembled apparatus.
FIG. 6 is a side elevation, of view of the assembled apparatus.
Description of Preferred Embodiments .
The apparatus illustrated in the drawings is shown to comprise as a top member a sample application plate 10 having a plurality of holes 12 extending therethrough into which a liquid sample can be placed.
The middle member is a membrane support plate 14 positioned beneath the plate 10 with the liquid .
' .
,: .. ;:: . ~-, . ... .
WO 91/02073 PCr/US90/0408~i t ~ ,., ., .
20,B3~
7 -} ;
permeable membrane 1~, when present, being placed between the two plates. The membrane support plate 14 has openlngs 18 into which the cannulas 20 are inserted. As shown, each cannula has a sidewall
top member, a middle member and a bottom member in a sandwiched relatl`onship. The top and middle members are plates having holes therethrough and between which a membrane can be placed. When the plates are placed one on top of another the holes are in axial alignment and the cross-sectional area o~ the holes at the hottom surface of the top plate is greater than the cross-sectional area of the holes at the top surface of the middle plate. ~urthermore, the sidewalls of the holes in the middle plate extend below the surface of the plate. As described later, the holes in the middle plate are preferably contained within tubes, herein termed cannulas, which are inserted through openings initially formed in the middle plate.
The bottom member of the apparatus is a collection chamber which has an opening on the upper side which faces the bottom surface of the middle plateO The chamber contains a port through one of its surfaces so that a YacUUm can be created within the chamber. The chamber contains means for accepting a microtiter plate containing a plurality of wells. In the assembled apparatus, the ends of the sidewalls of the holes which extend beneath the bottom surface of the middle plate are located within the wells of the microtiter plate. The apparatus contains means ~or securing the three members together in a vacuum type relationship when a membrane is positioned between the top and middle plates.
In use, the apparatus described above is first assembled with a liquid permeable membrane, to which a receptor can be bound, placed between the top and middle plates, and without the plate containing the wells being positioned in the bottom chamber. The membrane can either have the receptor already bound thereto or a solution containing the receptor can be . . . , : .
WO9l/0~073 PCr/US~0/0~086 ¦
9;8 6 added to the holes in the top plate. In the later instance, vacuum is then created in the chamber with, for example, a peristaltic pump, so that the solution can be pulled past the membrane at a highly controlled consistent rate.
Having the receptor now bound to the membrane, the liquid sample containiny the target ligand is added to the holes in the top plate. As above described, vacuum is applied and the liquid is drawn directly through the membrane, through the holes in the middle plate, and then discharged into the collection chamber. As the liquid sample passes through the membrane, the ligand is bound to the receptor on the membrane. Thereafter, a solution containing an enzyme conjugate which has binding specificity for the target ligand is drawn through the membrane and is, in turn, bound to the target ligand.
This step is then followed by a washing step to remove enzyme conjugate which did not bind to the ligand.
After the above described steps, the bottom chamber is separated from the top and middle plates, which remain secured together, and is emptied of any liquid which may be present from previous steps.
Then, the microtiter plate is positioned in the bottom chamber and the apparatus is reassembled with the sidewall extensions o~ the middle plate now extending into the wells of the microtiter plate.
A solution contaihing a substrate for the bound enzyme, which on reaction therewith gives a colored product, is then add~d ~o the holes in the top plate and drawn through the membrane, through the holes in the middle plate and, in turn, is deposited into the wells of the microtiter plate. Sufficient substrate solution is uti.lized so that on collection in the wells the solution extends above the bottom ends of the sidewall extensions. This feature is important '; ~'', ~ . , , ,, ,,,'j ~,' '. . ' ' ', I ' '' ' ' ~ .::' .: -' .
' ': ' , , ' , '. ' ' . ' .. .
., ' . ' .... ' ' ,' '' ": ' ' . ' ' , , WO~1/02073 l'CT/US9~/04086 ~; , ,390 in order to insure that the volume of solution transferred from the holes in the upper plate to the wells in the micr~ iter plate is the same for each set of holes and ~ells in the apparatus. In turn, this permits measurement of well to well differences in color intensity which correlate to differences in bound ligand. After collection is complete, the bottom chamber is again separated from the remaining structure, the microtiter plate removed from the chamber and the development of color of the solution in the wells determined.
Brief Description of the Drawln~s FIG. 1 is an exploded perspective view showing the apparatus of the present invention with certain parts omitted and with a membrane included.
FIG. Z is an exploded side elevation with certain parts broken away and shown in section.
FIG. 3 is a cross-section taken vertically through the apparatus.
FIG. 4 is an enlargement, partly in section, of one of the clamps.
FIG. 5 is a top plan view of the assembled apparatus.
FIG. 6 is a side elevation, of view of the assembled apparatus.
Description of Preferred Embodiments .
The apparatus illustrated in the drawings is shown to comprise as a top member a sample application plate 10 having a plurality of holes 12 extending therethrough into which a liquid sample can be placed.
The middle member is a membrane support plate 14 positioned beneath the plate 10 with the liquid .
' .
,: .. ;:: . ~-, . ... .
WO 91/02073 PCr/US90/0408~i t ~ ,., ., .
20,B3~
7 -} ;
permeable membrane 1~, when present, being placed between the two plates. The membrane support plate 14 has openlngs 18 into which the cannulas 20 are inserted. As shown, each cannula has a sidewall
5 portion 22 with a hole 24 extending through the cannula. As assembled, the holes 12 in the application plate 10 are in axial alignment with the holes 24 in the cannulas 20 located in the support plate 14. In order to attain registry of the two 10 plates 10 and 14 to achieve the aforementioned alignment, the support plate contains studs 26 which are adapted to fit into the holes 30 of the application plate. Four thumb screws 34 serve ~o secure the application plate 10 and membrane support 15 plate 14 together.
Turning to the cannulas 20, as mentioned, they contain a sidewall portion 22 and a hole 24. As shown in FIG. 3, the sidewalls 22 of the cannulas extend beneath the bottom surface of the support plate 14.
20 In addition, as illustrated, the sidewalls of the cannulas extend above the top surface of the middle plate, the top end of the cannulas containing a flanged portion 3~ which abuts against the membrane 16.
To permit easy application of liquid to the holes 12 in the application plate, the holes are conically shaped and, as illustrated, the cross-sectional area of the holes 12 at the bottom surface of the plate 10 is greater than the cross-sectional area of the holes 24 at the ~op surface of the cannulas 20. The cross-sectional area of the ~langed portion 36 of the cannulas 20 is greater than the cross-sectional area of the holes 12 at the bottom surface of the application plat:e. These features, in combination, assuro that liquid applied in the holes 12 will be . .
W091tO~073 PCT/US90/04086
Turning to the cannulas 20, as mentioned, they contain a sidewall portion 22 and a hole 24. As shown in FIG. 3, the sidewalls 22 of the cannulas extend beneath the bottom surface of the support plate 14.
20 In addition, as illustrated, the sidewalls of the cannulas extend above the top surface of the middle plate, the top end of the cannulas containing a flanged portion 3~ which abuts against the membrane 16.
To permit easy application of liquid to the holes 12 in the application plate, the holes are conically shaped and, as illustrated, the cross-sectional area of the holes 12 at the bottom surface of the plate 10 is greater than the cross-sectional area of the holes 24 at the ~op surface of the cannulas 20. The cross-sectional area of the ~langed portion 36 of the cannulas 20 is greater than the cross-sectional area of the holes 12 at the bottom surface of the application plat:e. These features, in combination, assuro that liquid applied in the holes 12 will be . .
W091tO~073 PCT/US90/04086
6 8 drawn through the membrane 16 and, in turn, through the holes 24 in the cannulas 20.
In order to assure that liquid, when being drawn through the membrane 16, does not disperse laterally and cause cross-contamination between samples, flexible "O" rings 38 are placed below the flanyed portion 36 o~ the cannulas between the flanged portions and the membrane support plate 14.
Alternatively, instead of the illustrated individual "O" rings, other gas~.eting means can be used to prevent lateral dispersion of liquid through the membrane. Another example is a flexibls perforated diaphragm, having dimensions co-extensive with the array of holes in the top plate, which can be disposed on either or both sides of the flanged portions of the cannulas, a diaphragm on both sides being preferred.
Referring still to the drawings, the illustrated apparatus is shown to contain, as a bottom member, a collection chamber 40 for receiving liquid drawn through the application plate 10, the membrane 16 and the holes 24 in the support plate 14. T~ receive liquid, the collection chamber is open on the upper side thereof which faces the bottom surface of the membrane support plate.
To enable vacuum to be created within the chamber to draw liquid through the apparatus, the chamber contains the vacuum port 42 which is attached via tubing and valving to a vacuum pump such as a peristaltic pump. As illustrated in FIGS. 2 and 3, the bottom internal surface 44 of the collection chamber 40 is sloped to permit fluid to flow to the ~acuum port and, in turn, be removed from the chamber 40. The chamber 40 also contains a port 46 which contains a valve tnot shown) to relieve vacuum in the chamber after each sample solution (i.e., receptor, ligand, etc.) has been drawn through the membrane. By . . , -,,; :
WO91/02073 Pr/US~0~04086 J r~
so doing, the succeeding solution placed in all of the holes of the application plate can be drawn simultaneously through the men~rane by vacuum created by the peristaltic pump.
As indicated above, the final step in the assay procedure described herein involves collectiny the colored liquid reaction product of substrate and enzyme so that color can be measured and, in turn, the - amount of bound target ligand determined. To that end, there is provided for insertion into the chamber a microtiter plate 50 containing a plurality of wells 52 for collection of liquid. ~s illustrated, the lower portion of the microtiter plate contains a skirt 54 which, when the plate is inserted into the chamber ~0, rests upon the ledge 56 in the chamber. In order to permit the vacuum created in the chamber 40 to communicate with the upper and middle portions of the apparatus and thereby permit drawing of the liquid through the apparatus, the ledge 56 is, as indicated, discontinuous between points 58 and 60. Thus, when a vacuum is drawn in the chamber 40, the microtiter plate does not form a vacuum tight seal with the ledge 56.
- As shown in FIG. 3, when the microtiter plate 50 is contained within the collection chamber 40, the end of the sidewall extension of the cannulas 20 is located within the wells 52 o~ the microtiter plate 50. The extent to which the end of the cannulas so extend into the wells is such that when the assay ~procedure is completed and the colored reaction product of substrate and enzyme has been drawn into the wells 52, the end of the cannulas are submerged below the surface of the liquid. By so doing, when the membrane support plate and, in turn, the cannulas are withdrawn from the wells, the reservoir of liquid in the wells prevents drops from being retained on the .
~ ~ .
WO 91/02073 ~'cr/US90/O'tO86 C~,Q~! ,' - ,1~'`, 10 ends of the cannulas, which drops may vary in volume from cannula to cannula. If drops were permitted to remain on the cannulas, the amount of solution transferred from the cannulas into the wells would be different, from well to well, and precise quantitation of bound ligand, from well to well, would not be obtainable.
The collection chamber is secured in vacuum tight relationship to the membrane support plate 14 by means of conventional clampin~ and gasketing. An example of such is shown in the drawings to include clamps 62 and gasket 48. Turning specifically to the clamps 62, they are shown in FIG. 4 to include a lever 64, a bracket 66 which is secured to the collection chamber 40 by means of the screws 68, a bracket 70 which is secured to the membrane support plate by means of the screws 72, and a tension screw 74 adjustably mounted in the housing 76. The housing 76 is pivotally mounted at the base of the lever 64 which, in turn, is pivotally mounted on the bracket 66. The upper bracket 70 is slotted to accommodate the head of the screw 74.
To clamp the chamber 40 to the support plate 14 using the clamps 62, the head of the screw 74 is inserted into the slot of the bracket 70 by pivoting the sc:rew into position. Then, the lever 64 is raised, thus forcing the screw and, in turn, the bracket 70 downward to, in combination with the gasket 48, to e~festively seal the chamber to the membrane support plate. By adjusting the screw within the housing 76, the tension can he adjusted to insure a vacuum seal.
The following example illustrates the use o~ the above described apparatus. All parts of the apparatus, except for the thumb screws and clamps, were made of plastic and a peristaltic pump was used .
,., :: , ~ ~
WO91/02()73 PCr/~S~0/040~6 11 ,''.~.2,0C3986 to create vacuum. A commercially available nitrocellulose membrane sold for use in immunoassays was employed. Prior to insertion between the sample application plate and the support plate, the membrane was wetted with distilled water. The apparatus when assembled measured about 6-1/2" x 4-1~2" and was approximately 2-1/2" high. 96 conically shaped holes were present in the application plate. The membrane support plates contained 96 cannulas as illustrated.
A commercially availab]e microtiter plate with 96 wells was used. The procedure employed was as-follows.
100 ~1 of distilled water was placed into each of the 96 holes in the sample application plate. To expel air from the system, the distilled water was drawn by vacuum through the apparatus into the collection chamber and removed through the vacuum port. When the holes in the application plate are emptied of liquid and air first contacts the membrane, no further flow of liquid or air can occur because of capillarity. Thus, throughout the procedure, there remains a continuous liquid phase between the lower surface of the membrane and the bottom of the cannulas.
200 ~1 of human serum albumin (HSA) solution (50 ~g/ml in Tris buffer~ was then placed in each of the holes in the application plate. The peristaltic vacuum pump was started and over a period of five minutes the HSA solution was drawn through the apparatus and discharged in~o the collection chamber.
The ~acuum relief valve was then spened and again closed after which time 200 ~1 of a 3% bovine serum albumin (BSA) solution was added to each of the holes in the application plate. Over a five minute period, the BSA solution was drawn through the membrane and discharged into the collection chamber, this step .,,....,,; . , , . . . , . : . .,, ' . ... .
.. .. . . ,: .
W09l/0~073 PC~/US90/(~086 ~ q ~ S 12 serving to block the vacant ~inding sites on the membrane. Again, the vacuum relief valve wac; opened and closed. Next, 200 ~1 of a lolO00 dilution (in Tris buffer) of mouse derived anti-HSA was deposited into each of the holes in the application plate and drawn through` the membrane and discharged into the chamber over a five minute period. After again opening and closing the vacuum relief valve, 200 ~1 of a 1:1000 dilution of goat derived anti-mouse IgG
alkaline phosphatase conjugate solution was deposited into each of the holes in the application plat:e and over a five minute period drawn through the apparatus and discharged into the collection chamber. After again opening and closing the vacuum relief valve, unbo~md conjugate was removed from the membrane by washing the mem~rane three times with 200 ~1 of t.~ris buffer drawn through the membrane in thirty second intervals.
Subsequent to the foregoing washing step, the vacuum relief valve was opened and the collection chamber separated from the top assembly oc the apparatus containing the application plate~ membrane and membrane support plate. The chamber was emptied of any liquid remaining from the foregoing operations and the top assembly was placed on a piece of paper toweling to remove any excess liquid off the bottom of the cannulas. A commercially available 96 well flal~
bottomed microtiter plate was placed in the collection chamber and the apparatus reassembled by clampinq the collection chamher and the membrane support plate together. As so assembled, the bottom of the cannulas extend into the wells of the microtiter plate. After closing the vacuum relief valve, 200 ~1 of a 0.5 mg/ml p-nitrophenyl phosphate solution was added to the wells and drawn through the apparatus over a five minute period. The colored solution produced by the W091/02073 PCr/US90/040X6 f~ 2l~6g9~86:, , reaction of substrate and enzyme was collected in the wells of the microtiter plate with the solution extending above the end of the cannulas positioned in the wells.
After the above procedure was completed, vacuum was relieved and the chamber was again separated from the membrane support plate. The cannulas were slowly withdrawn from the wells so that there was no remaining liquid on the exterior of the cannulas. The microtiter plate was then removed from the chamber and the color in each well read using a commercially available automatic ELISA plate reader. The amount of color determined in each well was substantially th~
same indicating the precision of the assay since equal quantities of mouse derived anti-HSA (the target ligand~ were applied to each hole in the application plate.
~ hile, in the foregoing procedure, a standard known concentration of target ligand was employed, an unknown amount of target ligand can be identified by using the above protocol. In this instance, instead of one standard known concentration of target ligand being smployed, an entire range of known concentrations or target ligands are utilized, thereby yielding plotted data which correlates color development with the known concentrations of target ligands in a standard curve. The standard curve is then used to translate ~he color development of an unknown concentration of target ligand to its actual conce~ntration. In practice, unknown samples can fill a number of the holes in the application plate and the balance of the holes can contain the standard solutions for construction of the standard curve.
Although the protocol illustrated as an example above is commonly called a direct sandwich assay, the apparatus and methodology herein described is not i :
r WV91/02073 ~cr/~ls~ 4~
39~
1~
limited to this type of assay. It can also be used in direct assays, indirect assays, indirect sand~ich assays, competitive assayci, etc. Essentially any immunoassay arrangement used with standard ELISA type techniques can bè mimicked with the apparatus and methodology described herei.n.
In order to assure that liquid, when being drawn through the membrane 16, does not disperse laterally and cause cross-contamination between samples, flexible "O" rings 38 are placed below the flanyed portion 36 o~ the cannulas between the flanged portions and the membrane support plate 14.
Alternatively, instead of the illustrated individual "O" rings, other gas~.eting means can be used to prevent lateral dispersion of liquid through the membrane. Another example is a flexibls perforated diaphragm, having dimensions co-extensive with the array of holes in the top plate, which can be disposed on either or both sides of the flanged portions of the cannulas, a diaphragm on both sides being preferred.
Referring still to the drawings, the illustrated apparatus is shown to contain, as a bottom member, a collection chamber 40 for receiving liquid drawn through the application plate 10, the membrane 16 and the holes 24 in the support plate 14. T~ receive liquid, the collection chamber is open on the upper side thereof which faces the bottom surface of the membrane support plate.
To enable vacuum to be created within the chamber to draw liquid through the apparatus, the chamber contains the vacuum port 42 which is attached via tubing and valving to a vacuum pump such as a peristaltic pump. As illustrated in FIGS. 2 and 3, the bottom internal surface 44 of the collection chamber 40 is sloped to permit fluid to flow to the ~acuum port and, in turn, be removed from the chamber 40. The chamber 40 also contains a port 46 which contains a valve tnot shown) to relieve vacuum in the chamber after each sample solution (i.e., receptor, ligand, etc.) has been drawn through the membrane. By . . , -,,; :
WO91/02073 Pr/US~0~04086 J r~
so doing, the succeeding solution placed in all of the holes of the application plate can be drawn simultaneously through the men~rane by vacuum created by the peristaltic pump.
As indicated above, the final step in the assay procedure described herein involves collectiny the colored liquid reaction product of substrate and enzyme so that color can be measured and, in turn, the - amount of bound target ligand determined. To that end, there is provided for insertion into the chamber a microtiter plate 50 containing a plurality of wells 52 for collection of liquid. ~s illustrated, the lower portion of the microtiter plate contains a skirt 54 which, when the plate is inserted into the chamber ~0, rests upon the ledge 56 in the chamber. In order to permit the vacuum created in the chamber 40 to communicate with the upper and middle portions of the apparatus and thereby permit drawing of the liquid through the apparatus, the ledge 56 is, as indicated, discontinuous between points 58 and 60. Thus, when a vacuum is drawn in the chamber 40, the microtiter plate does not form a vacuum tight seal with the ledge 56.
- As shown in FIG. 3, when the microtiter plate 50 is contained within the collection chamber 40, the end of the sidewall extension of the cannulas 20 is located within the wells 52 o~ the microtiter plate 50. The extent to which the end of the cannulas so extend into the wells is such that when the assay ~procedure is completed and the colored reaction product of substrate and enzyme has been drawn into the wells 52, the end of the cannulas are submerged below the surface of the liquid. By so doing, when the membrane support plate and, in turn, the cannulas are withdrawn from the wells, the reservoir of liquid in the wells prevents drops from being retained on the .
~ ~ .
WO 91/02073 ~'cr/US90/O'tO86 C~,Q~! ,' - ,1~'`, 10 ends of the cannulas, which drops may vary in volume from cannula to cannula. If drops were permitted to remain on the cannulas, the amount of solution transferred from the cannulas into the wells would be different, from well to well, and precise quantitation of bound ligand, from well to well, would not be obtainable.
The collection chamber is secured in vacuum tight relationship to the membrane support plate 14 by means of conventional clampin~ and gasketing. An example of such is shown in the drawings to include clamps 62 and gasket 48. Turning specifically to the clamps 62, they are shown in FIG. 4 to include a lever 64, a bracket 66 which is secured to the collection chamber 40 by means of the screws 68, a bracket 70 which is secured to the membrane support plate by means of the screws 72, and a tension screw 74 adjustably mounted in the housing 76. The housing 76 is pivotally mounted at the base of the lever 64 which, in turn, is pivotally mounted on the bracket 66. The upper bracket 70 is slotted to accommodate the head of the screw 74.
To clamp the chamber 40 to the support plate 14 using the clamps 62, the head of the screw 74 is inserted into the slot of the bracket 70 by pivoting the sc:rew into position. Then, the lever 64 is raised, thus forcing the screw and, in turn, the bracket 70 downward to, in combination with the gasket 48, to e~festively seal the chamber to the membrane support plate. By adjusting the screw within the housing 76, the tension can he adjusted to insure a vacuum seal.
The following example illustrates the use o~ the above described apparatus. All parts of the apparatus, except for the thumb screws and clamps, were made of plastic and a peristaltic pump was used .
,., :: , ~ ~
WO91/02()73 PCr/~S~0/040~6 11 ,''.~.2,0C3986 to create vacuum. A commercially available nitrocellulose membrane sold for use in immunoassays was employed. Prior to insertion between the sample application plate and the support plate, the membrane was wetted with distilled water. The apparatus when assembled measured about 6-1/2" x 4-1~2" and was approximately 2-1/2" high. 96 conically shaped holes were present in the application plate. The membrane support plates contained 96 cannulas as illustrated.
A commercially availab]e microtiter plate with 96 wells was used. The procedure employed was as-follows.
100 ~1 of distilled water was placed into each of the 96 holes in the sample application plate. To expel air from the system, the distilled water was drawn by vacuum through the apparatus into the collection chamber and removed through the vacuum port. When the holes in the application plate are emptied of liquid and air first contacts the membrane, no further flow of liquid or air can occur because of capillarity. Thus, throughout the procedure, there remains a continuous liquid phase between the lower surface of the membrane and the bottom of the cannulas.
200 ~1 of human serum albumin (HSA) solution (50 ~g/ml in Tris buffer~ was then placed in each of the holes in the application plate. The peristaltic vacuum pump was started and over a period of five minutes the HSA solution was drawn through the apparatus and discharged in~o the collection chamber.
The ~acuum relief valve was then spened and again closed after which time 200 ~1 of a 3% bovine serum albumin (BSA) solution was added to each of the holes in the application plate. Over a five minute period, the BSA solution was drawn through the membrane and discharged into the collection chamber, this step .,,....,,; . , , . . . , . : . .,, ' . ... .
.. .. . . ,: .
W09l/0~073 PC~/US90/(~086 ~ q ~ S 12 serving to block the vacant ~inding sites on the membrane. Again, the vacuum relief valve wac; opened and closed. Next, 200 ~1 of a lolO00 dilution (in Tris buffer) of mouse derived anti-HSA was deposited into each of the holes in the application plate and drawn through` the membrane and discharged into the chamber over a five minute period. After again opening and closing the vacuum relief valve, 200 ~1 of a 1:1000 dilution of goat derived anti-mouse IgG
alkaline phosphatase conjugate solution was deposited into each of the holes in the application plat:e and over a five minute period drawn through the apparatus and discharged into the collection chamber. After again opening and closing the vacuum relief valve, unbo~md conjugate was removed from the membrane by washing the mem~rane three times with 200 ~1 of t.~ris buffer drawn through the membrane in thirty second intervals.
Subsequent to the foregoing washing step, the vacuum relief valve was opened and the collection chamber separated from the top assembly oc the apparatus containing the application plate~ membrane and membrane support plate. The chamber was emptied of any liquid remaining from the foregoing operations and the top assembly was placed on a piece of paper toweling to remove any excess liquid off the bottom of the cannulas. A commercially available 96 well flal~
bottomed microtiter plate was placed in the collection chamber and the apparatus reassembled by clampinq the collection chamher and the membrane support plate together. As so assembled, the bottom of the cannulas extend into the wells of the microtiter plate. After closing the vacuum relief valve, 200 ~1 of a 0.5 mg/ml p-nitrophenyl phosphate solution was added to the wells and drawn through the apparatus over a five minute period. The colored solution produced by the W091/02073 PCr/US90/040X6 f~ 2l~6g9~86:, , reaction of substrate and enzyme was collected in the wells of the microtiter plate with the solution extending above the end of the cannulas positioned in the wells.
After the above procedure was completed, vacuum was relieved and the chamber was again separated from the membrane support plate. The cannulas were slowly withdrawn from the wells so that there was no remaining liquid on the exterior of the cannulas. The microtiter plate was then removed from the chamber and the color in each well read using a commercially available automatic ELISA plate reader. The amount of color determined in each well was substantially th~
same indicating the precision of the assay since equal quantities of mouse derived anti-HSA (the target ligand~ were applied to each hole in the application plate.
~ hile, in the foregoing procedure, a standard known concentration of target ligand was employed, an unknown amount of target ligand can be identified by using the above protocol. In this instance, instead of one standard known concentration of target ligand being smployed, an entire range of known concentrations or target ligands are utilized, thereby yielding plotted data which correlates color development with the known concentrations of target ligands in a standard curve. The standard curve is then used to translate ~he color development of an unknown concentration of target ligand to its actual conce~ntration. In practice, unknown samples can fill a number of the holes in the application plate and the balance of the holes can contain the standard solutions for construction of the standard curve.
Although the protocol illustrated as an example above is commonly called a direct sandwich assay, the apparatus and methodology herein described is not i :
r WV91/02073 ~cr/~ls~ 4~
39~
1~
limited to this type of assay. It can also be used in direct assays, indirect assays, indirect sand~ich assays, competitive assayci, etc. Essentially any immunoassay arrangement used with standard ELISA type techniques can bè mimicked with the apparatus and methodology described herei.n.
Claims (9)
1. An apparatus for use in a ligand-receptor assay procedure for the quantitative determination of the concentration of a target ligand in a liquid sample, said apparatus comprising a top member, a middle member, and a bottom member in a sandwiched relationship, said top and middle members being plates having a plurality of holes therethrough with said holes in the two plates being in axial alignment and with the cross-sectional area of the holes at the bottom surface of the top plate being greater than the cross-sectional area of the holes at the top surface of the middle plate, the holes in said middle plate being further characterized in that the sidewalls thereof extend below the bottom surface of the middle plate, said bottom member being a chamber having an opening on the upper side thereof facing the bottom surface of said middle plate, said chamber containing (1) at least one port extending through a surface thereof to permit a vacuum to be created within said chamber and (2) means for accepting a microtiter plate containing a plurality of wells such that, when the microtiter plate is positioned with the chamber, the ends of the holes extending beneath the bottom surface of the middle plate are located within said wells, said apparatus containing means for securing said three members together in vacuum tight relationship such that when (1) a liquid permeable membrane is placed between the top and middle plates, (2) liquid is placed in the holes in the top plate and (3) vacuum is created in the chamber, the liquid is drawn at a controlled rate directly through the membrane without lateral dispersion, through the holes in the middle plate and, in turn, into the chamber.
2. The apparatus of claim 1 wherein a liquid permeable membrane is positioned between said top and middle plates, said membrane being capable of binding the receptor utilized in the assay procedure.
3. The apparatus of claim 1 wherein the microtiter plate is positioned within the chamber.
4. The apparatus of claim 1 wherein the holes in the middle plate are contained within cannulas inserted into the middle plate.
5. The apparatus of claim 4 wherein the sidewalls of the cannulas extend above the top surface of the middle plate, the top end of the cannulas containing a flanged portion.
6. The apparatus of claim 5 wherein a liquid permeable membrane is positioned between said top and middle plates, said membrane being capable of binding the receptor utilized in the assay procedure.
7. The apparatus of claim 6 wherein the microtiter plate is positioned within the chamber.
8. The apparatus of claim 7 wherein the membrane is nitrocellulose.
9. A process for accomplishing a ligand-receptor assay procedure utilizing the apparatus described in claim 1 which has positioned between said top and middle plates a liquid permeable membrane having a receptor bound thereto, said process comprising (1) adding a solution containing a ligand to said holes in the top plate and, by means of vacuum created in said chamber, drawing said solution through the membrane, whereby said ligand is bound to said receptor, (2) adding a solution, containing an enzyme conjugate which has bonding specificity for the ligand, to said holes in the top plate, and by means of vacuum created in said chamber, drawing said solution through the membrane, whereby the conjugate is bound to the ligand, (3) removing conjugate which did not bind to the ligand from the membrane, (4) inserting a microtiter plate into the chamber of the apparatus, (5) adding a solution which contains a substrate for the bound enzyme, which on reaction therewith gives a colored product, to said holes in the top plate and, by means of vacuum created in said chamber, drawing said solution through the membrane, through said holes in the middle plate and, in turn, into the wells in the microtiter plate, said amount of said solution drawn through said membrane in step (5) being such that the solution in the wells extends above the bottom ends of the holes extending beneath the surface of the middle plate, and (6) removing the microtiter plate from the apparatus and reading the amount of color of the solution contained in the wells.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US38622689A | 1989-07-28 | 1989-07-28 | |
| US386,226 | 1989-07-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2063986A1 true CA2063986A1 (en) | 1991-01-29 |
Family
ID=23524692
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2063986 Abandoned CA2063986A1 (en) | 1989-07-28 | 1990-07-20 | Method and apparatus for rapid immunoassays |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU6141690A (en) |
| CA (1) | CA2063986A1 (en) |
| WO (1) | WO1991002073A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4107262A1 (en) * | 1991-03-07 | 1992-09-10 | Eppendorf Geraetebau Netheler | SUCTION DEVICE FOR MEMBRANE MICROTITER PLATES |
| WO1999019067A1 (en) | 1997-10-10 | 1999-04-22 | Biosepra, Inc. | Aligned multiwell multiplate stack and method for processing biological/chemical samples using the same |
| AU6054099A (en) * | 1999-09-16 | 2001-04-17 | Immunetics, Inc. | Membrane immunoassays for detection of multiple tick-borne diseases |
| WO2009112952A2 (en) * | 2008-03-12 | 2009-09-17 | Cellectricon Ab | Apparatus and method for tip alignment in multiwell plates |
| US8057754B2 (en) | 2008-03-12 | 2011-11-15 | Cellectricon Ab | Apparatus and method for tip alignment in multiwell plates |
| ES2358699B1 (en) | 2011-03-09 | 2012-03-14 | Zf Biotox, S.L. | MICROPLACE FOR BIOLOGICAL TESTS. |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4031197A (en) * | 1973-04-25 | 1977-06-21 | Gte New Ventures Corporation | In vitro method for determining allergic hypersensitivity |
| US4427415A (en) * | 1979-01-05 | 1984-01-24 | Cleveland Patrick H | Manifold vacuum biochemical test method and device |
| US4761378A (en) * | 1983-03-04 | 1988-08-02 | American Home Products Corp. (Del.) | Microbiological testing apparatus |
| US4626509A (en) * | 1983-07-11 | 1986-12-02 | Data Packaging Corp. | Culture media transfer assembly |
| US4599315A (en) * | 1983-09-13 | 1986-07-08 | University Of California Regents | Microdroplet test apparatus |
| DE3407849A1 (en) * | 1984-02-29 | 1985-08-29 | Alois 3201 Algermissen Höft | METHOD AND DEVICE FOR SIMULTANEOUSLY APPLYING A VARIETY OF LIQUID SAMPLES TO A SLIDE |
| IE850830L (en) * | 1985-04-01 | 1986-10-01 | Noctech Ltd | Enzyme immunoassay method |
| NL8600056A (en) * | 1986-01-13 | 1987-08-03 | Nederlanden Staat | METHOD FOR DRAWING A MEMBER OF AN IMMUNOLOGICAL COUPLE, METHOD FOR PREPARING A CARRIER TO WHICH A MEMBER OF AN IMMUNOLOGICAL COUPLE IS BONDED AND SUITABLE FOR ANALYSIS FOR CARRYING OUT SUCH METHODS |
| US4895706A (en) * | 1986-10-28 | 1990-01-23 | Costar Corporation | Multi-well filter strip and composite assemblies |
| US4834946A (en) * | 1987-02-05 | 1989-05-30 | Levin Andrew E | Apparatus for blot screening numerous, small volume, antibody solutions |
-
1990
- 1990-07-20 CA CA 2063986 patent/CA2063986A1/en not_active Abandoned
- 1990-07-20 AU AU61416/90A patent/AU6141690A/en not_active Abandoned
- 1990-07-20 WO PCT/US1990/004086 patent/WO1991002073A1/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| WO1991002073A1 (en) | 1991-02-21 |
| AU6141690A (en) | 1991-03-11 |
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