CA2158340A1 - Enhanced microtitre plate and immunoassays conducted therein - Google Patents
Enhanced microtitre plate and immunoassays conducted thereinInfo
- Publication number
- CA2158340A1 CA2158340A1 CA 2158340 CA2158340A CA2158340A1 CA 2158340 A1 CA2158340 A1 CA 2158340A1 CA 2158340 CA2158340 CA 2158340 CA 2158340 A CA2158340 A CA 2158340A CA 2158340 A1 CA2158340 A1 CA 2158340A1
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- Prior art keywords
- reflective
- wells
- walls
- coating
- microtitre plate
- Prior art date
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- 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
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
- B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Clinical Laboratory Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optical Measuring Cells (AREA)
Abstract
A microtitre plate containing a plurality of wells for conducting immunoassays wherein one or more of the sides, bottom and lid walls of each well are formed with a reflective, metallized surface. The surface may be an inner coating on the walls of clear or opaque plates, or an outer coating on the walls of clear plates. Alternatively, the plate may be formed, for example, by stamping from a reflective metal material. Improved sensitivity in photometric detection results from photon emitting immunoassays conducted in the plate.
Description
~WO 94Q1379 ,~ ~ 5 8 3 ~ O PCT/CA94/00158 n~R~r~ED MICRO-l-.L~ PI~ATE AND I r JN~ C-e~YS ~ NL~u~
2 ~ ~ ~ I N ~
3 FIELD OF THE lNv~ ON
4 This invention relates to microtitre plates and ;mml,nogenic assays conducted therein.
6 R~KG~OUND OF THE l~v~-.. ~ON
7 Microtitre plates are moulded plastic plates or 8 trays having a plurality of depressions or wells in which 9 chemical and biological tests are carried out. Tmmllnogenic assays involving an antibody-antigen reaction to detect an 11 antibody or antigen in biological materials such as serum 12 are most commoPly performed in microtitre plates.
13 Colorimetric immunoassays are widely used, the most common 14 type being an ELISA (enzyme-linked immunosorbent assay) method. This technique is an enzyme immunoassay used for 16 the detection of antibodies or antigens. Antigens are 17 immobilized on the surfaces of the wells of the microtitre 18 plates, following which, the sample to be assayed for 19 particular antibodies, typically serum, is added to the wells in specific assay dilutions. This step is followed by 21 the immuno detection of the antibody/antigen complexes, 22 usually by photometrically measuring an enzymatically 23 labelled colour-forming immune complex. In colorimetric 24 ;mmllnoassays~ a source of incident light is directed onto or through the assay reagents (typically in a clear multi-well 26 microtitre plate). A portion of the radiant energy of the 27 incident beam is absorbed by a component the assay. The 28 light which is transmitted from the assay is detected by a 29 suitable photometer to provide a measurement of the concentration of a component of the assay. Colorimetric 31 immunoassays are photon absorbing assays.
32 Other photometric- assay techniques performed in 33 microtitre plates are termed photon emitting immunoassays.
34 These include chemiluminescent, biolum~inescent and fluorescent ;mmllnoassays. In these assays photons are 36 emitted, for example as a result of the action of an enzyme, 37 or through the use of particular photon emitting labels.
WO94/21379 21~ 8 3 ~ O PCT/CA94/00158 1 Emitted photons are detected by suitable photometric devices 2 such as luminometers or fluorometers.
3 Immunoassays are typically conducted in microtitre 4 plates. Such plates are typically made of a light transmitting plastic polymer such as polystyrene or 6 polyvinylchoride. It is usually important that the 7 microtitre plate be formed from a light transmitting plastic 8 since reading of the assay results is typically done through 9 the contents in the wells, particularly for colorimetric immunoassays.
11 In the case of photon emitting ;mmllnoassays, the 12 microtitre plates may be made of opaque plastic, such as 13 black or white polystyrene, in order to reduce "cross-talk'~
14 in photometrically reading the results from well to well (i.e. to reduce interference caused by stray photons).
16 While cross-talk may be lessened by such opaque microtitre 17 plates, the sensitivity of the results is lessened. For 18 instance, black plates absorb some of the photons being 19 emitted from the plates while white plates simply scatter the photons.
21 Japanese Patent 61-215947 teaches that the optical 22 interference in colorimetric ;mmllnoassays carried out in 23 transparent microtitre plates can be reduced by constructing 24 wells optically independently with an opaque layer on the well walls.
26 An ;mmllnoassay technique of greater 27 sensitivity often resorted to is a radio;mml~noassay (RIA).
28 However, this technique is costly both in time and equipment 29 needs as it involves the use of radioactive materials.
These materials require special handling and licenses from 31 the time they are ordered until they are safely stored at a 32 radioactive waste site. However, even with the drawbacks, 33 the high degree of sensitivity provided by this technique 34 for some assays (i.e. where the amount of material being assayed is very small or where the volume of sample is very 36 small) makes it an indispensable diagnostic tool. If these 37 levels of sensitivity could be reached by a non-isotopic 2~834~ - -1 method, then costs could be dramatically lowere~ ana a~m~c~-2 safer work environment could be achieved.
3 ~here is a need for a microtitre plate which 4 provides for improved sensitivity in photometric immllnoassay techniques, particularly or photon emitting 7 mml~noassays 6 such as, chemiluminescent, bioluminescent and fluorescent 7 ;mmllnoassays.
8 S~MM~Y OF T~E l~VL.. ~ lON
9 This invention overcomes the above mentioned difficulties by providing a microtitre plate with a 11 multiplicity of wells or depressions with walls that are 12 highly reflective. The plates may be formed with a 13 reflective metal, ceramic or semiconductor coating on one or 14 both of the side or bottom walls of the wells.
Alternatively, the plates may be formed from reflective 16 metal. Preferred reflective metals, ceramics or semi-17 conductors for coatings include (in a highly re~lective 18 form) aluminum, tin, magnesium, zinc, cadmium, indium, 19 transition metals such as silver, chromium, gold, platinum and nic~el, silicon, germanium, silica and alumina or alloys 21 cont~i n; ng at least one of these metals. The plates may 22 also be stamped from a highly reflective metal, for example, 23 from bright aluminum foil.
24 The invention also provides an improved method of conducting a photon emitting immllnoassay in the reflective 26 plates. ~nh~nced sensitivity results from the use of the 27 reflective plates.
28 Broadly stated the invention provides a microtitre 29 plate comprising a plate member having top and bottom surfaces, the top surface defining a plurality of spaced, 31 upwardly opening wells, each of said wells having side 32 and/or bottom walls with inner and outer surface, and one or 33 both of the side and bottom walls of said wells providing a 34 reflective, metallized surface.
~ AMEN~E0SffE~r WO94/21379 2 ~ 5 8 3 ~ 0 PCT/CA94/00158 1 The invention also provides an improved method of 2 conducting a photon emitting immunoassay in a multi-well 3 microtitre plate, wherein an antigen-antibody complex is 4 formed in the wells and photons emitted from the wells are detected by a photometric device. The improvement comprises 6 conducting the immunoassay in a microtitre plate having a 7 reflective metallized surface on one or both of the side or 8 bottom walls of the wells to reflect photons emitted from 9 the wells into the photometric device.
It should be understood that the term "wells~, as 11 used herein and in the claims, in association with 12 microtitre plates, is meant to include both depressions 13 having only a bottom wall (ex. shallow, round bottom 14 depressions), and generally cylindrical wells, having both side and bottom walls. Generally cylindrical wells 16 typically have round, flat or conical bottom walls. The 17 side walls are typically vertical, but could vary, for 18 example as conical or fluted side walls.
19 It should also be understood that the term "walls"
as used herein and in the claims is meant to include the 21 inner or outer surfaces of the wells, regardless of the well 22 shape. Thus, the term wall, when used in association with 23 a depression shaped well, refers to its rounded surface, 24 although the depression does not in fact possess discrete side and bottom walls.
26 The phrase ~reflective, metallized", as used 27 herein and in the claims, is meant to include highly 28 reflective surfaces produced from shiny metallic materials, 29 for instance, al~lm;n-~m, tin, transition metals, ceramics and semiconductors (all in a highly reflective form), as 31 distinct from surfaces that are merely light scattering due 32 to glassy or glossy properties.
33 n~.c~TPTIoN OF THE ~K~K~ E~
34 The microtitre plates in accordance with the present invention are preferably produced by coating 36 stAn~rd multi-well microtitre plates with a reflective, -1 metallized coating on at least a portion of the inner or 2 outer side or bottom walls of the plates. Alternatively, 3 the plates according to this invention are formed by 4 stamping ~he wells into a reflective metal, such as bright aluminum foil.
6 The reflective metallized coatings are preferably 7 metal coatings because they are highly reflective and 8 inexpensive to apply. However, ceramic or semiconductor 9 materials may be applied as highly reflective coatings in assay applications ~m~n~;ng an inert surface or a surface 11 with particular immobilization ~ualities.
12 If a clear,transparent microtitre plate is used, 13 the coating may be provided on the inner side and/or bottom 14 walls of the plates. Alternatively, with a transparent microtitre plate, the outer side and/or bottom walls of the plates are coated. If an opaque, coloured plate is used, 17 the coating is provided on the inner side and/or bottom ~8 walls. The particular areas coated will vary with the 19 particular assay technique and photometric plate reading e~7i~ment which is to be used. For instance, ~or bottom 21 read plates, the bottoms of the wells may be left uncoated.
22 Bottom read plates may include a reflective lid or plate 23 cover for use in the actual rea~;ng equipment. Top read 24 plates, which are most commonly used, are preferably coated on both the bottom and side walls to maximize the advantages 26 of the reflective coating.
27 The walls of the microtitre plates may be shaped 28 to ,m~i m; ze the utility of the reflective coating. For 29 example, the walls may be curved (ex. rounded or parabolic) or facetted to maximize upward reflections of photons into 31 the photometric detecting equipment.
32 State of the art coating techniques for example 33 physical vapour deposition, chemical vapour deposition or 34 electroless deposition, may be used. Physical vapour ~MENOEI~ S~tEET
,, WO94/2l37g 2 ~ 4 0 PCT/CA94/00158 1 deposition techni~ues include sputtering, magnetron 2 sputtering, vacuum deposition and ion plating.
3 The coating is applied to a thickness such that 4 the walls of the plate are no longer transparent to light, but are highly reflective of light.
6 Microtitre plates which are coated on the outside 7 walls with the metallized reflective coating are preferred 8 to m;n;m;ze immobilization problems encountered in the assay 9 techniques. An outer reflective coating preserves the inner wall surface (typically polystyrene) for which most 11 immunoassay techniques have been designed. If the inner 12 walls are coated with the metallized reflective coating, the 13 assay technique may be altered to overcome any 14 immobilization problems. Alternatively, a clear, inner coating of a plastic such as polystyrene may be provided on 16 the walls themselves to avoid toxicity or immobilization 17 problems with the metal coating. Reflective ceramic or 18 semiconductor coatings may also be advantageously used to 19 overcome toxicity or immobilization problems.
Shiny metals can be used for the reflective 21 metallized coating, for example, all~m;nnm, tin, magnesium, 22 zinc, cadmium, indium, transition metals such as silver, 23 nickel, gold, platinum and chromium or alloys of these or 24 other metals. Thin reflective coatings of ceramics, such as alumina and silica, or semiconductors such as silicon and 26 geranium, may also be used. Alnm;nllm is particularly 27 preferred since it does not interfere significantly with the 28 assay techni~ues. The particular coating material or alloy 29 chosen will vary with the assay technique to be performed in the wells.
31 Tm~llnoaSSay techniques are well known in the art.
32 The metallized reflective plates of the present invention 33 are advantageously used in colorimetric immunoassays such as 34 ELISA, to ~nh~nce the sensitivity of the readings off the plates. However, the plates are particulary useful in 36 photon emitting ;mmllnoassays, including chemilllm;n~cent, 37 biolllm;nescent and fluorescent immunoassays. Such assays ~ WO94/21379 215 ~3~ ~ PCT/CA94/00158 1 are described in detail in the literature, see ~or example, 2 L.J. Kricka and T.J.N. Carter, Clinical and Biochemical 3 Luminescence, Marcel Dekker, New York (1982)i L.J.Kricka et 4 al., Analytical Applications of Bioluminescence and Chemiluminescence, Academic Press, New York (1984)i and N.
6 Monji and A. Castro, Conjunction of Haptens and 7 Macromolecules to Phycobili Protein for Application in 8 Florescence Tmm~lnoassay~ Reviews on Tmmllnoassay Technology, 9 S.B.Pal (ed), Vol 1, ChA~m~n and Hall, New York (1988).
If the plates used in photon emitting assays have a 11 metallized reflective coating on the outer walls of the 12 otherwise transparent plates, the assays may be conducted 13 without alteration. However, if the coating is on the inner 14 walls of the plate, and immobilization or toxicity problems are encountered with the metal coating, the assay technigue 16 should be modified to include a first step of coating the 17 inner walls with an a&erent plastic film. A reflective 18 coating of ceramic or semiconductor material may also be 19 used. These latter coatings might also be applied, in a clear form, over the reflective coating.
21 The invention is further illustrated by the 22 following non-limiting examples.
23 Example 1 24 This example illustrates the preparation of a metallized microtitre plate in accordance with the present 26 invention. A stAn~Ard clear flat bottomed polystyrene 27 microtitre plate (obtained from Corning) was coated by 28 magnetron sputtering with all~m;nl~m metal as follows:
29 Magnetron Sputtering Conditions:
Eguipment - Perkin Elmer 4410 31 Target - 99.999% Alllm;nllm 32 Power - lkW
33 Time - 20 min 34 R.P.M. - 3 Coating Thickness - 4000 Angstroms 36 Base Pressure - 2 X 10-7 Torr 37 Cathode/Substrate Distance - 65 mm 2 ~ 0 1 Working Gas - Argon 2 Working Gas Pressure - 20 mT
3 The plates were oriented on the substrate table 4 such that either the insides of the individual wells and the top side of the plate or the outsides of the individual 6 wells and the bottom side of the top of plates were 7 metallized (line of sight coating)~. The coating thickness 8 on the flat portions of the plates coated at normal 9 incidence was 4000 Angstroms.~`On the inside vertical walls of the wells, the coating thickness varied from 4000 11 Angstroms at the top to about 500 Angstroms at the bottom 12 (the alllm;nl~m coating was not transparent at the bottom).
13 Example 2 14 The two plates coated in accordance with the procedure set forth in Example 1 (i.e. metallized reflective 16 coating on the inside or outside side and bottom walls of 17 the wells) were tested both biologically and physically to 18 ~mo~qtrate utility for ;mmllnoassays and to evaluate for 19 reflectivity.
a) Reflectivity Tests 21 The reflectivity of the each of the plates was 22 compared to the reflectivity of stAn~Ard clear, white and 23 black microtitre plates using a Nanospec AFT reflectometer.
24 The instrument was stAn~Ardized against an aluminized optically flat silicon wafer (coated under the conditions 26 set forth in Example 1). Data was collected as a percentage 27 of the stAn~Ard. The results were as follows:
28 St~n~Ard 100%
29 Plate, metallized inner walls 98 Plate, metallized outer walls 96~
31 Plate, clear polystyrene 9.0%
32 Plate, white polystyrene 7.5 33 Plate, black polystyrene 4.0~
34 The metallized plates in accordance with the 3S present invention were clearly much more reflective than the 36 clear or opa~ue, coloured plates. This is in part due to 37 the lack of scattering and absorption of light in the ~ W094/21379 2 15 8 3 4 ~ PCT/CA94/00158 1 metallized plates relative to the clear and opaque, coloured 2 plates. This demonstrates that in any photon emitting 3 assay, the metallized plates will result in a greater 4 capture of emitted photons since photons not directed at the detector initially will be redirected with m;n;m~l loss, 7 6 even after multiple reflections within the plate.
7 b) Biological Tests 8 A unit of alkaline phosphatase enzyme was serially 9 diluted to extinction (from 10 to lO9). Samples from dilutions were placed in individual wells of a metallized ll microtitre dish (coated as in Example l, inner side and 12 bottom walls) and a normal clear polystyrene microtitre 13 dish. All wells were precoated with bovine serum albumin to 14 m;n;m; ze enzyme deactivation through adsorption. The microtitre dishes were then placed into a photon counting 16 camera chamber and exposed to the enzymes substrate, p-17 nitrophenyl phosphate. Photons from the action of the 18 enzyme on the substrate were counted if they reached the l9 camera lens system. Emitted photons were counted from both plates simultaneously. It was found that the metallized 21 plates resulted in the detection of lO times more photons 22 than did the non-metallized plate. This suggests that 23 l~lm;n~ccent/florescent types of assays will be at least lO
24 times as sensitive or re~uire lO times less sample if the metallized plate is used.
26 All publications mentioned in this specification 27 are indicative of the level of skill of those skilled in the 28 art to which this invention pertains. All publications are 29 herein incorporated by reference to the same extent as if each individual publication was specifically and 3l individually indicated to be incorporated by reference.
32 Although the foregoing invention has been 33 described in some detail by way of illustration and example 34 for purposes of clarity of underst~n~;ng, it will be obvious that certain changes and modifications may be practised ' 36 within the scope of the appended claims.
6 R~KG~OUND OF THE l~v~-.. ~ON
7 Microtitre plates are moulded plastic plates or 8 trays having a plurality of depressions or wells in which 9 chemical and biological tests are carried out. Tmmllnogenic assays involving an antibody-antigen reaction to detect an 11 antibody or antigen in biological materials such as serum 12 are most commoPly performed in microtitre plates.
13 Colorimetric immunoassays are widely used, the most common 14 type being an ELISA (enzyme-linked immunosorbent assay) method. This technique is an enzyme immunoassay used for 16 the detection of antibodies or antigens. Antigens are 17 immobilized on the surfaces of the wells of the microtitre 18 plates, following which, the sample to be assayed for 19 particular antibodies, typically serum, is added to the wells in specific assay dilutions. This step is followed by 21 the immuno detection of the antibody/antigen complexes, 22 usually by photometrically measuring an enzymatically 23 labelled colour-forming immune complex. In colorimetric 24 ;mmllnoassays~ a source of incident light is directed onto or through the assay reagents (typically in a clear multi-well 26 microtitre plate). A portion of the radiant energy of the 27 incident beam is absorbed by a component the assay. The 28 light which is transmitted from the assay is detected by a 29 suitable photometer to provide a measurement of the concentration of a component of the assay. Colorimetric 31 immunoassays are photon absorbing assays.
32 Other photometric- assay techniques performed in 33 microtitre plates are termed photon emitting immunoassays.
34 These include chemiluminescent, biolum~inescent and fluorescent ;mmllnoassays. In these assays photons are 36 emitted, for example as a result of the action of an enzyme, 37 or through the use of particular photon emitting labels.
WO94/21379 21~ 8 3 ~ O PCT/CA94/00158 1 Emitted photons are detected by suitable photometric devices 2 such as luminometers or fluorometers.
3 Immunoassays are typically conducted in microtitre 4 plates. Such plates are typically made of a light transmitting plastic polymer such as polystyrene or 6 polyvinylchoride. It is usually important that the 7 microtitre plate be formed from a light transmitting plastic 8 since reading of the assay results is typically done through 9 the contents in the wells, particularly for colorimetric immunoassays.
11 In the case of photon emitting ;mmllnoassays, the 12 microtitre plates may be made of opaque plastic, such as 13 black or white polystyrene, in order to reduce "cross-talk'~
14 in photometrically reading the results from well to well (i.e. to reduce interference caused by stray photons).
16 While cross-talk may be lessened by such opaque microtitre 17 plates, the sensitivity of the results is lessened. For 18 instance, black plates absorb some of the photons being 19 emitted from the plates while white plates simply scatter the photons.
21 Japanese Patent 61-215947 teaches that the optical 22 interference in colorimetric ;mmllnoassays carried out in 23 transparent microtitre plates can be reduced by constructing 24 wells optically independently with an opaque layer on the well walls.
26 An ;mmllnoassay technique of greater 27 sensitivity often resorted to is a radio;mml~noassay (RIA).
28 However, this technique is costly both in time and equipment 29 needs as it involves the use of radioactive materials.
These materials require special handling and licenses from 31 the time they are ordered until they are safely stored at a 32 radioactive waste site. However, even with the drawbacks, 33 the high degree of sensitivity provided by this technique 34 for some assays (i.e. where the amount of material being assayed is very small or where the volume of sample is very 36 small) makes it an indispensable diagnostic tool. If these 37 levels of sensitivity could be reached by a non-isotopic 2~834~ - -1 method, then costs could be dramatically lowere~ ana a~m~c~-2 safer work environment could be achieved.
3 ~here is a need for a microtitre plate which 4 provides for improved sensitivity in photometric immllnoassay techniques, particularly or photon emitting 7 mml~noassays 6 such as, chemiluminescent, bioluminescent and fluorescent 7 ;mmllnoassays.
8 S~MM~Y OF T~E l~VL.. ~ lON
9 This invention overcomes the above mentioned difficulties by providing a microtitre plate with a 11 multiplicity of wells or depressions with walls that are 12 highly reflective. The plates may be formed with a 13 reflective metal, ceramic or semiconductor coating on one or 14 both of the side or bottom walls of the wells.
Alternatively, the plates may be formed from reflective 16 metal. Preferred reflective metals, ceramics or semi-17 conductors for coatings include (in a highly re~lective 18 form) aluminum, tin, magnesium, zinc, cadmium, indium, 19 transition metals such as silver, chromium, gold, platinum and nic~el, silicon, germanium, silica and alumina or alloys 21 cont~i n; ng at least one of these metals. The plates may 22 also be stamped from a highly reflective metal, for example, 23 from bright aluminum foil.
24 The invention also provides an improved method of conducting a photon emitting immllnoassay in the reflective 26 plates. ~nh~nced sensitivity results from the use of the 27 reflective plates.
28 Broadly stated the invention provides a microtitre 29 plate comprising a plate member having top and bottom surfaces, the top surface defining a plurality of spaced, 31 upwardly opening wells, each of said wells having side 32 and/or bottom walls with inner and outer surface, and one or 33 both of the side and bottom walls of said wells providing a 34 reflective, metallized surface.
~ AMEN~E0SffE~r WO94/21379 2 ~ 5 8 3 ~ 0 PCT/CA94/00158 1 The invention also provides an improved method of 2 conducting a photon emitting immunoassay in a multi-well 3 microtitre plate, wherein an antigen-antibody complex is 4 formed in the wells and photons emitted from the wells are detected by a photometric device. The improvement comprises 6 conducting the immunoassay in a microtitre plate having a 7 reflective metallized surface on one or both of the side or 8 bottom walls of the wells to reflect photons emitted from 9 the wells into the photometric device.
It should be understood that the term "wells~, as 11 used herein and in the claims, in association with 12 microtitre plates, is meant to include both depressions 13 having only a bottom wall (ex. shallow, round bottom 14 depressions), and generally cylindrical wells, having both side and bottom walls. Generally cylindrical wells 16 typically have round, flat or conical bottom walls. The 17 side walls are typically vertical, but could vary, for 18 example as conical or fluted side walls.
19 It should also be understood that the term "walls"
as used herein and in the claims is meant to include the 21 inner or outer surfaces of the wells, regardless of the well 22 shape. Thus, the term wall, when used in association with 23 a depression shaped well, refers to its rounded surface, 24 although the depression does not in fact possess discrete side and bottom walls.
26 The phrase ~reflective, metallized", as used 27 herein and in the claims, is meant to include highly 28 reflective surfaces produced from shiny metallic materials, 29 for instance, al~lm;n-~m, tin, transition metals, ceramics and semiconductors (all in a highly reflective form), as 31 distinct from surfaces that are merely light scattering due 32 to glassy or glossy properties.
33 n~.c~TPTIoN OF THE ~K~K~ E~
34 The microtitre plates in accordance with the present invention are preferably produced by coating 36 stAn~rd multi-well microtitre plates with a reflective, -1 metallized coating on at least a portion of the inner or 2 outer side or bottom walls of the plates. Alternatively, 3 the plates according to this invention are formed by 4 stamping ~he wells into a reflective metal, such as bright aluminum foil.
6 The reflective metallized coatings are preferably 7 metal coatings because they are highly reflective and 8 inexpensive to apply. However, ceramic or semiconductor 9 materials may be applied as highly reflective coatings in assay applications ~m~n~;ng an inert surface or a surface 11 with particular immobilization ~ualities.
12 If a clear,transparent microtitre plate is used, 13 the coating may be provided on the inner side and/or bottom 14 walls of the plates. Alternatively, with a transparent microtitre plate, the outer side and/or bottom walls of the plates are coated. If an opaque, coloured plate is used, 17 the coating is provided on the inner side and/or bottom ~8 walls. The particular areas coated will vary with the 19 particular assay technique and photometric plate reading e~7i~ment which is to be used. For instance, ~or bottom 21 read plates, the bottoms of the wells may be left uncoated.
22 Bottom read plates may include a reflective lid or plate 23 cover for use in the actual rea~;ng equipment. Top read 24 plates, which are most commonly used, are preferably coated on both the bottom and side walls to maximize the advantages 26 of the reflective coating.
27 The walls of the microtitre plates may be shaped 28 to ,m~i m; ze the utility of the reflective coating. For 29 example, the walls may be curved (ex. rounded or parabolic) or facetted to maximize upward reflections of photons into 31 the photometric detecting equipment.
32 State of the art coating techniques for example 33 physical vapour deposition, chemical vapour deposition or 34 electroless deposition, may be used. Physical vapour ~MENOEI~ S~tEET
,, WO94/2l37g 2 ~ 4 0 PCT/CA94/00158 1 deposition techni~ues include sputtering, magnetron 2 sputtering, vacuum deposition and ion plating.
3 The coating is applied to a thickness such that 4 the walls of the plate are no longer transparent to light, but are highly reflective of light.
6 Microtitre plates which are coated on the outside 7 walls with the metallized reflective coating are preferred 8 to m;n;m;ze immobilization problems encountered in the assay 9 techniques. An outer reflective coating preserves the inner wall surface (typically polystyrene) for which most 11 immunoassay techniques have been designed. If the inner 12 walls are coated with the metallized reflective coating, the 13 assay technique may be altered to overcome any 14 immobilization problems. Alternatively, a clear, inner coating of a plastic such as polystyrene may be provided on 16 the walls themselves to avoid toxicity or immobilization 17 problems with the metal coating. Reflective ceramic or 18 semiconductor coatings may also be advantageously used to 19 overcome toxicity or immobilization problems.
Shiny metals can be used for the reflective 21 metallized coating, for example, all~m;nnm, tin, magnesium, 22 zinc, cadmium, indium, transition metals such as silver, 23 nickel, gold, platinum and chromium or alloys of these or 24 other metals. Thin reflective coatings of ceramics, such as alumina and silica, or semiconductors such as silicon and 26 geranium, may also be used. Alnm;nllm is particularly 27 preferred since it does not interfere significantly with the 28 assay techni~ues. The particular coating material or alloy 29 chosen will vary with the assay technique to be performed in the wells.
31 Tm~llnoaSSay techniques are well known in the art.
32 The metallized reflective plates of the present invention 33 are advantageously used in colorimetric immunoassays such as 34 ELISA, to ~nh~nce the sensitivity of the readings off the plates. However, the plates are particulary useful in 36 photon emitting ;mmllnoassays, including chemilllm;n~cent, 37 biolllm;nescent and fluorescent immunoassays. Such assays ~ WO94/21379 215 ~3~ ~ PCT/CA94/00158 1 are described in detail in the literature, see ~or example, 2 L.J. Kricka and T.J.N. Carter, Clinical and Biochemical 3 Luminescence, Marcel Dekker, New York (1982)i L.J.Kricka et 4 al., Analytical Applications of Bioluminescence and Chemiluminescence, Academic Press, New York (1984)i and N.
6 Monji and A. Castro, Conjunction of Haptens and 7 Macromolecules to Phycobili Protein for Application in 8 Florescence Tmm~lnoassay~ Reviews on Tmmllnoassay Technology, 9 S.B.Pal (ed), Vol 1, ChA~m~n and Hall, New York (1988).
If the plates used in photon emitting assays have a 11 metallized reflective coating on the outer walls of the 12 otherwise transparent plates, the assays may be conducted 13 without alteration. However, if the coating is on the inner 14 walls of the plate, and immobilization or toxicity problems are encountered with the metal coating, the assay technigue 16 should be modified to include a first step of coating the 17 inner walls with an a&erent plastic film. A reflective 18 coating of ceramic or semiconductor material may also be 19 used. These latter coatings might also be applied, in a clear form, over the reflective coating.
21 The invention is further illustrated by the 22 following non-limiting examples.
23 Example 1 24 This example illustrates the preparation of a metallized microtitre plate in accordance with the present 26 invention. A stAn~Ard clear flat bottomed polystyrene 27 microtitre plate (obtained from Corning) was coated by 28 magnetron sputtering with all~m;nl~m metal as follows:
29 Magnetron Sputtering Conditions:
Eguipment - Perkin Elmer 4410 31 Target - 99.999% Alllm;nllm 32 Power - lkW
33 Time - 20 min 34 R.P.M. - 3 Coating Thickness - 4000 Angstroms 36 Base Pressure - 2 X 10-7 Torr 37 Cathode/Substrate Distance - 65 mm 2 ~ 0 1 Working Gas - Argon 2 Working Gas Pressure - 20 mT
3 The plates were oriented on the substrate table 4 such that either the insides of the individual wells and the top side of the plate or the outsides of the individual 6 wells and the bottom side of the top of plates were 7 metallized (line of sight coating)~. The coating thickness 8 on the flat portions of the plates coated at normal 9 incidence was 4000 Angstroms.~`On the inside vertical walls of the wells, the coating thickness varied from 4000 11 Angstroms at the top to about 500 Angstroms at the bottom 12 (the alllm;nl~m coating was not transparent at the bottom).
13 Example 2 14 The two plates coated in accordance with the procedure set forth in Example 1 (i.e. metallized reflective 16 coating on the inside or outside side and bottom walls of 17 the wells) were tested both biologically and physically to 18 ~mo~qtrate utility for ;mmllnoassays and to evaluate for 19 reflectivity.
a) Reflectivity Tests 21 The reflectivity of the each of the plates was 22 compared to the reflectivity of stAn~Ard clear, white and 23 black microtitre plates using a Nanospec AFT reflectometer.
24 The instrument was stAn~Ardized against an aluminized optically flat silicon wafer (coated under the conditions 26 set forth in Example 1). Data was collected as a percentage 27 of the stAn~Ard. The results were as follows:
28 St~n~Ard 100%
29 Plate, metallized inner walls 98 Plate, metallized outer walls 96~
31 Plate, clear polystyrene 9.0%
32 Plate, white polystyrene 7.5 33 Plate, black polystyrene 4.0~
34 The metallized plates in accordance with the 3S present invention were clearly much more reflective than the 36 clear or opa~ue, coloured plates. This is in part due to 37 the lack of scattering and absorption of light in the ~ W094/21379 2 15 8 3 4 ~ PCT/CA94/00158 1 metallized plates relative to the clear and opaque, coloured 2 plates. This demonstrates that in any photon emitting 3 assay, the metallized plates will result in a greater 4 capture of emitted photons since photons not directed at the detector initially will be redirected with m;n;m~l loss, 7 6 even after multiple reflections within the plate.
7 b) Biological Tests 8 A unit of alkaline phosphatase enzyme was serially 9 diluted to extinction (from 10 to lO9). Samples from dilutions were placed in individual wells of a metallized ll microtitre dish (coated as in Example l, inner side and 12 bottom walls) and a normal clear polystyrene microtitre 13 dish. All wells were precoated with bovine serum albumin to 14 m;n;m; ze enzyme deactivation through adsorption. The microtitre dishes were then placed into a photon counting 16 camera chamber and exposed to the enzymes substrate, p-17 nitrophenyl phosphate. Photons from the action of the 18 enzyme on the substrate were counted if they reached the l9 camera lens system. Emitted photons were counted from both plates simultaneously. It was found that the metallized 21 plates resulted in the detection of lO times more photons 22 than did the non-metallized plate. This suggests that 23 l~lm;n~ccent/florescent types of assays will be at least lO
24 times as sensitive or re~uire lO times less sample if the metallized plate is used.
26 All publications mentioned in this specification 27 are indicative of the level of skill of those skilled in the 28 art to which this invention pertains. All publications are 29 herein incorporated by reference to the same extent as if each individual publication was specifically and 3l individually indicated to be incorporated by reference.
32 Although the foregoing invention has been 33 described in some detail by way of illustration and example 34 for purposes of clarity of underst~n~;ng, it will be obvious that certain changes and modifications may be practised ' 36 within the scope of the appended claims.
Claims (11)
1. A microtitre plate comprising:
a plate member having top and bottom surfaces, the top surface defining a plurality of spaced, upwardly opening wells integral with the top surface, each of the wells having side and/or bottom walls with inner and outer surfaces; and the bottom walls, and optionally the side walls, of the wells providing a reflective, metallized surface on the inner surface of the walls.
a plate member having top and bottom surfaces, the top surface defining a plurality of spaced, upwardly opening wells integral with the top surface, each of the wells having side and/or bottom walls with inner and outer surfaces; and the bottom walls, and optionally the side walls, of the wells providing a reflective, metallized surface on the inner surface of the walls.
2. The microtitre plate as set forth in claim 1, wherein the wells are formed from a reflective metallized material or are provided with a reflective metal coating on the inner surface of the well walls.
3. The microtitre plate as set forth in claim 1, wherein the wells are formed from a clear material and a reflective metallized coating is provided on the inner surface of the well walls.
4. The microtitre plate as set forth in claim 1, wherein the wells are formed from an opaque material and a reflective metallized coating is provided on the inner surface of the well walls.
5. The microtitre plate as set forth in claim 2, wherein the reflective metallized coating is selected from the group consisting of reflective aluminum, tin, magnesium, zinc, cadmium, indium, transition metals, ceramics and semiconductors, or alloys containing at least one of these metals.
6. The microtitre plate as set forth in claim 2, wherein the metal material or coating is reflective aluminum.
7. The microtitre plate as set forth in claim 3, wherein the coating is reflective aluminum.
8. The microtitre plate as set forth in claim 4, wherein the coating is reflective aluminum.
9. In a method of conducting a photon emitting immunoassay in a multi-well microtitre plate, wherein an antigen-antibody complex is formed in the wells and photons emitted from the wells are detected by a photometric device, the improvement comprising:
conducting the immunoassay in a microtitre plate having a top surface defining a plurality of spaced upwardly opening wells and having a reflective metallized surface on the inner surface of the bottom walls, and optionally on the inner surface of the side walls, of the wells to reflect photons emitted from the wells into the photometric device.
conducting the immunoassay in a microtitre plate having a top surface defining a plurality of spaced upwardly opening wells and having a reflective metallized surface on the inner surface of the bottom walls, and optionally on the inner surface of the side walls, of the wells to reflect photons emitted from the wells into the photometric device.
10. The method as set forth in claim 9, wherein the reflective surface is a reflective metallized coating on the inner surface of the well walls, said coating being selected from the group consisting of reflective aluminum, tin, magnesium, zinc, cadmium, indium, transition metals, ceramics and semiconductors.
11. The method as set forth in claim 10, wherein the reflective surface is an aluminum reflective surface on the inner surface of the well walls.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US3204593A | 1993-03-16 | 1993-03-16 | |
| US08/032,045 | 1993-03-16 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2158340A1 true CA2158340A1 (en) | 1994-09-29 |
Family
ID=21862802
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2158340 Abandoned CA2158340A1 (en) | 1993-03-16 | 1994-03-11 | Enhanced microtitre plate and immunoassays conducted therein |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU6256194A (en) |
| CA (1) | CA2158340A1 (en) |
| WO (1) | WO1994021379A1 (en) |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5792426A (en) * | 1994-10-11 | 1998-08-11 | Schweizerische Eidgenossenschaft Vertreten Durch Das Ac-Laboratorium Spiez Der Gruppe Rustung | Multi-well titerplate for instrumental analysis |
| GB2319836B (en) * | 1996-11-25 | 2001-04-04 | Porvair Plc | Microplates |
| US6027695A (en) * | 1998-04-01 | 2000-02-22 | Dupont Pharmaceuticals Company | Apparatus for holding small volumes of liquids |
| WO1999049974A1 (en) * | 1998-04-01 | 1999-10-07 | The Du Pont Merck Pharmaceutical Company | Plate apparatus for holding small volumes of liquids |
| DE19903576C2 (en) * | 1999-01-29 | 2001-02-22 | Bodenseewerk Perkin Elmer Co | Quantitative determination of analytes in a heterogeneous system |
| DE10060560A1 (en) * | 2000-05-26 | 2001-12-06 | Bruker Optik Gmbh | Microtiter plate used for toxicological testing of pharmaceutically active ingredients and cosmetics, comprises a plate body with passages, and an infra red-permeable base formed on one side of the plate body |
| US6800491B2 (en) | 2001-06-08 | 2004-10-05 | Nalge Nunc International Corporation | Robotic reservoir without liquid hangup |
| WO2003000419A2 (en) * | 2001-06-21 | 2003-01-03 | Hybaid Limited | Sample well plate |
| GB0122286D0 (en) * | 2001-09-14 | 2001-11-07 | Scient Generics Ltd | Optical coatings for high-throughput laboratory consumables |
| US9005549B2 (en) * | 2003-01-17 | 2015-04-14 | Greiner Bio-One Gmbh | High throughput polymer-based microarray slide |
| DE10321472B4 (en) * | 2003-05-13 | 2005-05-12 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Fluidic module, used as multi-functional micro-reaction module for chemical reactions, has fluid zone between one side permeable to infrared and side with infrared reflective layer for on-line analysis |
| EP1529567A3 (en) * | 2003-11-07 | 2005-05-25 | Herbener, Heinz-Gerd | Sample holder with reaction chamber |
| EP2098293B1 (en) | 2008-02-22 | 2013-04-24 | Qiagen Instruments AG | Microtitre plate |
| EP3092474B1 (en) | 2014-06-12 | 2020-04-22 | Axion Biosystems, Inc. | Multiwell microelectrode array with optical stimulation |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6266141A (en) * | 1985-09-19 | 1987-03-25 | Sumitomo Bakelite Co Ltd | Vessel for fluorescent immunological measurement |
| SE8802282D0 (en) * | 1988-06-17 | 1988-06-17 | Wallac Oy | SAMPLE PLATE LIQUID SCINTILLATION COUNTER |
| US5082628A (en) * | 1989-09-19 | 1992-01-21 | Park Pharmaceuticals, Inc. | Luminometer |
| JPH05203562A (en) * | 1992-01-28 | 1993-08-10 | Hitachi Chem Co Ltd | Photoemission reactor and photometry using it |
| US5329123A (en) * | 1992-09-23 | 1994-07-12 | Wallac Oy | Method and a device for preventing optical crosstalk between liquid scintillation samples deposited on transculcent sample plates |
| US5298753A (en) * | 1992-11-12 | 1994-03-29 | Wallac Oy | Arrangement for counting liquid scintillation samples on bottom-window multi-well sample plates |
-
1994
- 1994-03-11 WO PCT/CA1994/000158 patent/WO1994021379A1/en active Application Filing
- 1994-03-11 AU AU62561/94A patent/AU6256194A/en not_active Abandoned
- 1994-03-11 CA CA 2158340 patent/CA2158340A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| AU6256194A (en) | 1994-10-11 |
| WO1994021379A1 (en) | 1994-09-29 |
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