CA2212193C - Method of detecting biological materials using a polyvinylidene fluoride membrane - Google Patents
Method of detecting biological materials using a polyvinylidene fluoride membrane Download PDFInfo
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- CA2212193C CA2212193C CA002212193A CA2212193A CA2212193C CA 2212193 C CA2212193 C CA 2212193C CA 002212193 A CA002212193 A CA 002212193A CA 2212193 A CA2212193 A CA 2212193A CA 2212193 C CA2212193 C CA 2212193C
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- membrane
- temperature
- subjected
- biological material
- polyvinylidene fluoride
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- 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
- G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
- Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
- Y10T436/255—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.] including use of a solid sorbent, semipermeable membrane, or liquid extraction
Abstract
The present invention provides an improved method of detecting or transferring a biological material whereby a biological material is adhered to a membrane which is then contacted with a detecting reagent, wherein the improvement comprises utilizing a polyvinylidene fluoride membrane which has been subjected to a temperature of at least about 80°C
but less than the temperature at which the membrane softens and deforms for a time sufficient to reduce the ability of the detecting reagent to directly adhere to the membrane.
but less than the temperature at which the membrane softens and deforms for a time sufficient to reduce the ability of the detecting reagent to directly adhere to the membrane.
Description
WO 96!32643 PfT/LTS96104139 METHOD OF DETECTING BIOLOGICAL MATERIALS
USING A POLYVINYLIDENE FLUORIDE MEMBRANE
TECHrfICAL FIELD OF THE INVENTION
The present ~Lnvention relates to an improvement in a method of detecting biological materials, e.g., proteins, using a porous membrane. The improved method involves the use of a heat-treated polyvinylidene fluoride membrane: .
BAt~KGROUND OF THE INVENTION
Membranes are: used in a variety of methods for detecting or transferring biological materials, such as western blotting (for proteins), northern blotting (for RNA), and southern, blotting (for DNA). In these various detection or transfer methods,.the biological material of interest. adheres t.o the membrane, e.g., a polyvinylidene fluoride: membrane, which is then typically subjected to a detecting reagent, e.g., a dye or stain, which preferentially adheres to the biological material of interest so as to enable detection of the biological material. Many detecting reagents, however, also generally adhere, at least to some extent, to the porous membrane, particularly a polyvinylidene fluoride membrane, thereby increasing the background noise and decreasing the sensitivity or signal-to-noise ratio for detecting the biological material of interest.
Thus, there remains a need for improving those methods of detecting biological materials which utilize membranes in conjunction with detecting reagents, particularly by reducing the tendency of detecting reagents to adhere directly to such membranes. The present ,invention ~~rovides such an improvement through the use of a modified polyvinylidene fluoride membrane which exhibits reduced background noise. These and other objects <~nd advant<~ges of the present invention, as well as additional inve:ntive features, will be apparent from the description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
The present invention provides an improved method of detecting or tran~;ferring a biological material whereby a biological materiel is adhered to a membrane which is then contacted wit=h a detecting reagent, wherein the improvement comprises utilizing a polyvinylidene fluoride membranes which has been subjected to a temperature of at least about 80 °C but less than the temperature at which the membrane softe=ns and def orms for a time suf f icient to reduce i;.he ability of the detecting reagent to directly adhere to the membrane.
DESCRIPT7:ON OF THE PREFERRED EMBODIMENTS
The present :invention involves the use of a heat-treated polyvinyl:Ldene fluoride membrane in a method of detecting or tran:~ferring a biological material. In particular, the p~~esent invention provides for the use of a heat-'treated po:Lyvinylidene fluoride membrane in a method of detecting or transferring a biological material whereby a biological material is adhered to the membrane which is then cowtacted with a detecting reagent. Such detecting reagents include dyes, stains, and the like.
Suitable such methods include western blotting, northern blotting, and southern blotting. Western blotting is a method for detecting or transferring proteins and is generally described in Towbin et al., Proc. Natl. Acad. Sci. USA, 76, 4350-4354 (1979).
Northern blotting is a method for detecting or .
transferring RNA's and is generally described in Thomas, Proc. Natl. Acad. Sci. USA, 77, 5201-5205 (1980).
Southern blotting is a method for detecting or transferring DNA's and is generally described in Southern, J. Mol. Biol., 98, 503-517 (1975). These detection or transfer methods, as well as numerous variations thereon and other detection or transfer procedures utilizing membranes, particularly hydrophobic membranes such as polyvinylidene fluoride membranes, are well-known in the art.
The present invention has particular applicability to protein detection or transfer methods, such as western blotting, inasmuch as proteins readily adhere to polyvinylidene fluoride membranes. Indeed, non-heat-treated polyvinylidene fluoride membranes are commercially available as FluoroTransTM membranes (Pall Corporation, East Hills, New York) for use in western blotting procedures. The heat-treated polyvinylidene fluoride membrane is desirably utilized in place of a non-heat-treated polyvinylidene fluoride membrane in western blotting .procedures, as well as in any other such detection or transfer procedure wherein a non-heat-treated polyvinylidene fluoride membrane is or can be utilized to some extent.
The polyvinylidene fluoride membrane can be prepared in any suitable manner, e.g., by using the wet casting procedure described in U.S. Patent 4,340,479. Any suitable polyvinylidene fluoride may be used, such as Kynar~ 761 resin (Atochem, Philadelphia, Pennsylvania).
The polyvinyliden~e fluoride will typically have a molecular weight ~of at least about 5,000 daltons, preferably a molecular weight of at least about 10,000 daltons.
The polyvinylidene fluoride membrane can have any suitable porosity. The membrane will typically have a pore rating of about 10 ~cm or less, more typically about 1 ~Cm or less, e.g., about 0.01-1 ~Cm, and most typically about 0.50 ~m or .less. Preferably, the membrane has a pore rating of about 0.05-0.45 hum or, even more preferably, about 0.05-0.2 ~cm.
The polyviny.lidene fluoride membrane can be heat-treated, or annealed, in any suitable manner, desirably at a temperature and for a period of time sufficient to WO 96/32643 ~ PCT/I1S96/04139 reduce the ability of the detecting reagent to directly adhere to the membrane. In other words, the heat-treatment preferaY>ly results in a polyvinylidene fluoride membrans~ to which the detecting reagent is less likely to adhere as compared to the non-heat-treated polyvinylidene f luoridea membrane .
Preferably, t:he polyvinylidene fluoride membrane is heated to a temperature of at least about 80 °C for a time sufficient to achieve the desired reduced adherence of the detecting reagent to the membrane. Of course, the membrane should not be heated at so high a temperature that the membrane becomes soft and deforms, either under its own weight or due to tension from any mechanical means by which the: membrane is supported during the heating process. Typically, the upper temperature limit will be about 160 °C. The amount of time of heating will vary with the hearing temperature and nature of the membrane being heated. For example, small pieces of membrane in flat :sheet form which are in direct contact with a high temperature surface may require only a brief exposure, e.g., lEass than one minute, to heat, while a rolled membrane o:E several hundred linear meters may require many hour:a of heating at low temperature for the .membrane to reach a suitable equilibrium temperature.
Thus, the membrane is preferably subjected to a temperature of about 80 °C to about 160 °C for a suitable period of time, preferably for about 5 minutes to about 64 hours, with the desirable time period generally decreasing as the temperature is increased. Accordingly, the membrane is preferably subjected to about 80 °C for about 48 hours or more, more preferably about 64 hours or more. Similarly, the membrane is preferably subjected to about 160 °C for about 5 minutes or more, more preferably about 10 minutes or more.
Preferably, the polyvinylidene fluoride membrane is subjected to a temperature of about 80 °C to about 150 °C, more typically a temperature of about 100 °C to about 150 °C, far a suitable period of time, desirably about 32 hours or more. Mere preferably, the membrane is subjected to a temperature of about 120 °C to about 150 °C for a suitable period of time, desirably about 1C
USING A POLYVINYLIDENE FLUORIDE MEMBRANE
TECHrfICAL FIELD OF THE INVENTION
The present ~Lnvention relates to an improvement in a method of detecting biological materials, e.g., proteins, using a porous membrane. The improved method involves the use of a heat-treated polyvinylidene fluoride membrane: .
BAt~KGROUND OF THE INVENTION
Membranes are: used in a variety of methods for detecting or transferring biological materials, such as western blotting (for proteins), northern blotting (for RNA), and southern, blotting (for DNA). In these various detection or transfer methods,.the biological material of interest. adheres t.o the membrane, e.g., a polyvinylidene fluoride: membrane, which is then typically subjected to a detecting reagent, e.g., a dye or stain, which preferentially adheres to the biological material of interest so as to enable detection of the biological material. Many detecting reagents, however, also generally adhere, at least to some extent, to the porous membrane, particularly a polyvinylidene fluoride membrane, thereby increasing the background noise and decreasing the sensitivity or signal-to-noise ratio for detecting the biological material of interest.
Thus, there remains a need for improving those methods of detecting biological materials which utilize membranes in conjunction with detecting reagents, particularly by reducing the tendency of detecting reagents to adhere directly to such membranes. The present ,invention ~~rovides such an improvement through the use of a modified polyvinylidene fluoride membrane which exhibits reduced background noise. These and other objects <~nd advant<~ges of the present invention, as well as additional inve:ntive features, will be apparent from the description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
The present invention provides an improved method of detecting or tran~;ferring a biological material whereby a biological materiel is adhered to a membrane which is then contacted wit=h a detecting reagent, wherein the improvement comprises utilizing a polyvinylidene fluoride membranes which has been subjected to a temperature of at least about 80 °C but less than the temperature at which the membrane softe=ns and def orms for a time suf f icient to reduce i;.he ability of the detecting reagent to directly adhere to the membrane.
DESCRIPT7:ON OF THE PREFERRED EMBODIMENTS
The present :invention involves the use of a heat-treated polyvinyl:Ldene fluoride membrane in a method of detecting or tran:~ferring a biological material. In particular, the p~~esent invention provides for the use of a heat-'treated po:Lyvinylidene fluoride membrane in a method of detecting or transferring a biological material whereby a biological material is adhered to the membrane which is then cowtacted with a detecting reagent. Such detecting reagents include dyes, stains, and the like.
Suitable such methods include western blotting, northern blotting, and southern blotting. Western blotting is a method for detecting or transferring proteins and is generally described in Towbin et al., Proc. Natl. Acad. Sci. USA, 76, 4350-4354 (1979).
Northern blotting is a method for detecting or .
transferring RNA's and is generally described in Thomas, Proc. Natl. Acad. Sci. USA, 77, 5201-5205 (1980).
Southern blotting is a method for detecting or transferring DNA's and is generally described in Southern, J. Mol. Biol., 98, 503-517 (1975). These detection or transfer methods, as well as numerous variations thereon and other detection or transfer procedures utilizing membranes, particularly hydrophobic membranes such as polyvinylidene fluoride membranes, are well-known in the art.
The present invention has particular applicability to protein detection or transfer methods, such as western blotting, inasmuch as proteins readily adhere to polyvinylidene fluoride membranes. Indeed, non-heat-treated polyvinylidene fluoride membranes are commercially available as FluoroTransTM membranes (Pall Corporation, East Hills, New York) for use in western blotting procedures. The heat-treated polyvinylidene fluoride membrane is desirably utilized in place of a non-heat-treated polyvinylidene fluoride membrane in western blotting .procedures, as well as in any other such detection or transfer procedure wherein a non-heat-treated polyvinylidene fluoride membrane is or can be utilized to some extent.
The polyvinylidene fluoride membrane can be prepared in any suitable manner, e.g., by using the wet casting procedure described in U.S. Patent 4,340,479. Any suitable polyvinylidene fluoride may be used, such as Kynar~ 761 resin (Atochem, Philadelphia, Pennsylvania).
The polyvinyliden~e fluoride will typically have a molecular weight ~of at least about 5,000 daltons, preferably a molecular weight of at least about 10,000 daltons.
The polyvinylidene fluoride membrane can have any suitable porosity. The membrane will typically have a pore rating of about 10 ~cm or less, more typically about 1 ~Cm or less, e.g., about 0.01-1 ~Cm, and most typically about 0.50 ~m or .less. Preferably, the membrane has a pore rating of about 0.05-0.45 hum or, even more preferably, about 0.05-0.2 ~cm.
The polyviny.lidene fluoride membrane can be heat-treated, or annealed, in any suitable manner, desirably at a temperature and for a period of time sufficient to WO 96/32643 ~ PCT/I1S96/04139 reduce the ability of the detecting reagent to directly adhere to the membrane. In other words, the heat-treatment preferaY>ly results in a polyvinylidene fluoride membrans~ to which the detecting reagent is less likely to adhere as compared to the non-heat-treated polyvinylidene f luoridea membrane .
Preferably, t:he polyvinylidene fluoride membrane is heated to a temperature of at least about 80 °C for a time sufficient to achieve the desired reduced adherence of the detecting reagent to the membrane. Of course, the membrane should not be heated at so high a temperature that the membrane becomes soft and deforms, either under its own weight or due to tension from any mechanical means by which the: membrane is supported during the heating process. Typically, the upper temperature limit will be about 160 °C. The amount of time of heating will vary with the hearing temperature and nature of the membrane being heated. For example, small pieces of membrane in flat :sheet form which are in direct contact with a high temperature surface may require only a brief exposure, e.g., lEass than one minute, to heat, while a rolled membrane o:E several hundred linear meters may require many hour:a of heating at low temperature for the .membrane to reach a suitable equilibrium temperature.
Thus, the membrane is preferably subjected to a temperature of about 80 °C to about 160 °C for a suitable period of time, preferably for about 5 minutes to about 64 hours, with the desirable time period generally decreasing as the temperature is increased. Accordingly, the membrane is preferably subjected to about 80 °C for about 48 hours or more, more preferably about 64 hours or more. Similarly, the membrane is preferably subjected to about 160 °C for about 5 minutes or more, more preferably about 10 minutes or more.
Preferably, the polyvinylidene fluoride membrane is subjected to a temperature of about 80 °C to about 150 °C, more typically a temperature of about 100 °C to about 150 °C, far a suitable period of time, desirably about 32 hours or more. Mere preferably, the membrane is subjected to a temperature of about 120 °C to about 150 °C for a suitable period of time, desirably about 1C
5 hours or more, e.c~., about 48-72 hours. Most preferably, the membrane is subjected to a temperature of about 135 °C to about 145 °~~, e.g., about 140 °C, for a suitable period of time, desirably about 12 hours or more, more desirably about 24 hours or more, e.g., about 36-64 hours, especially about 48 hours.
The heat-treatment of the polyvinylidene fluoride membrane can be ei:fected without the membrane being restrained; however, the membrane preferably is dimensionally restrained during the heat-treatment so as to minimize or avoid dimensional changes in the membrane, e.g., sririnkage. Any suitable means can be used to dimensionally rest:rain,the membrane. For example, the membrane: can be p7.aced into a frame or can be wound onto a core or roll, preferably with an interleaved material, such as a fibrous nonwoven material, to prevent layer-to-layer contact of t:he membrane. Most preferably, the membrane: is heat-treated in roll form interleaved with a polyester fibrous nonwoven material.
The heat-treatment can be carried out by any suitable: means. for example, the polyvinylidene fluoride can be subjected t.o the aforesaid temperature by contacting the membrane with a heated surface.
Alternatively, the membrane can be subjected to the desired temperature by placing the membrane, preferably 3o in roll form, in a suitable oven, e.g., a circulating air oven.
The heat-treatment of polyvinylidene fluoride membranes is more fully described in U.S. Patents 5,1,96,508 and 5,198,505. Those patents also describe certain improvements in surface modifications which can be obtained (and are desirably exhibited by the polyvinylidene fluoride membranes in the context of the WO 96132643 PCT/~TS96/04139 present invention) by heat-treating polyvinylidene fluoride membranes, albeit without an appreciation of the surprisingly reduced tendency of detecting reagents to adhere to such membranes in the context of the present invention.
A comparison of polyvinylidene fluoride membranes before and after heat-treatment has led to the discovery that the heat-treatment reduces the surface area of the membrane (as determined by BET analysis). While not seeking' to be bound to any particular theory explaining the surprisingly reduced tendency of detecting reagents to adhere to a heat-treated polyvinylidene fluoride membrane in the context of the present invention, it is believed that the reduced tendency of detecting reagents to adhere to the heat-treated polyvinylidene fluoride membrane is at least in part the result of the reduced surface area of the heat-treated membrane. Detecting reagents apparently have a greater difficulty in adhering to the reduced-surface area polyvinylidene fluoride 2o membrane, while the ability of biological materials, such as proteins, to adhere to the polyvinylidene fluoride membrane is not significantly altered. Thus, the background noise level is decreased, while improving the sensitivity or signal-to-noise ratio of the overall process.
The following examples further illustrate the present invention and, of course, should not be construed as in any way limiting its scope.
Example 1 This example illustrates the superiority of the present inventive method utilizing a heat-treated polyvinylidene fluoride membrane as compared to the same method utilizing a non-heat-treated polyvinylidene fluoride membrane.
A western transfer of a standard test protein mixture from a ge.l to a membrane was carried out using a WO 9f»/32643 PCT/LTS96/~4139 non-heat-treated polyvinylidene fluoride membrane (specif:ically, FlixoroTrans~ from Pail Corporation) and the same membrane which had been subjected in roll form to about x.40 °C in an air circulating oven for about 48 hours (:including t:he heat-up and cool-down time for the oven). The general protein transfer protocol set forth in the product in:~ert for FluoroTransTM was followed, using amido black stain as the detecting reagent. The respectiva membranes were then evaluated with a densitometer.
The' background intensity level (i.e., noise level) for the non-heat-treated polyvinylidene fluoride membrane was nearly twice that for the heat-treated polyvinylidene fluoridsa membrane. Moreover, the ratio of the intensity I5 of the highest peak to the background level was about 1.5:1 far the non-heat-treated .polyvinylidene fluoride membrane and about: 2.8:1 for the heat-treated polyvinylidene fluoride membrane. Thus, the reduction in the bacl~:ground intensity level was accompanied by an improvement in the: signal-to-noise ratio of over 100%.
Example 2 This example further illustrates the superiority of the pre~.ent inventive method utilizing a heat-treated polyvinylidene fluoride membrane as compared to commercially available protein transfer membranes.
Dyed protein markers were electrophoresed and transferred to (a) a non-heat-treated polyvinylidene fluoride: membrane having a pore rating of 0.2 um (specifically, FluoroTransTM from Pall Corporation), (b) a heat-treated polyvinylidene fluoride membrane having a pore rating of 0.2 ~Cm (specifically, FluoroTransTM from Pall Corporation subjected in roll form to about 140 °C
in an ai.r circulating oven for about 48 hours (including the heat.-up and cool-down time for the oven)), (c) a similarly heat-treated polyvinylidene fluoride membrane having a pore rating of 0.45 ~Cm, (d) a commercially-available, competitive polyvinylidene fluoride protein-transfer membrane having a pore rating of 0.45 Vim, and a commerciall~~-available, competitive nitrocellulose membrane: having a pore rating of 0.45 ~cm.
A polyvinylidene fluoride capture membrane was placed behind eactl of the membranes being evaluated so as to capture any protein which passed through the evaluated membranes and to thereby detect any protein "burn through." The protein transfer was allowed to proceed for 24 riours,.and then the capture membranes were examined for the x~resence of proteins.
The non-heat-treated 0.2 ~m pore rated polyvinylidene fluoride membrane (sample a) exhibited no significant protein "burn through." Similarly, the heat-treated 0.2 ~,m pore rated polyvinylidene fluoride membrane, (sample b) exhibited no significant protein "burn through." 'These results demonstrate that the heat-treatment process does not adversely affect the excellent properties of pol~winylidene fluoride membranes of 2o relatively small pore rating as regards protein "burn through ., "
As regards the other membranes which were evaluated, the commercially-available competitive 0.45 ~cm pore rated polyvinylidene fluoride membrane (sample d) exhibited a small, but signif~:cant, amount of protein "burn through,"
while the competitive 0.45 ~m pore rated nitrocellulose membrane (sample ea) exhibited a high degree of protein "burn through. " 7:n contrast, the heat-treated 0 . 45 ~Cm pore rated polyvinylidene fluoride membrane (sample c) exhibited no significant protein "burn through." These results demonstrate that the present inventive method , results in at least an equivalent, if not lower, level of protein "burn through" as compared to methods utilizing similar pore-rated, commercially-available, competitive membranes. As a result, the present inventive method can be expected to re:ault in at least as good, if not better, protein retention,, and ultimately greater sensitivity, thin methods involving those commercially-available, competitive membranes which were evaluated herein.
While this invention has been described with an emphasis upon preferred embodiments, it will be obvious to those of ordinary skill in the art that variations of the preferred products and methods may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims.
The heat-treatment of the polyvinylidene fluoride membrane can be ei:fected without the membrane being restrained; however, the membrane preferably is dimensionally restrained during the heat-treatment so as to minimize or avoid dimensional changes in the membrane, e.g., sririnkage. Any suitable means can be used to dimensionally rest:rain,the membrane. For example, the membrane: can be p7.aced into a frame or can be wound onto a core or roll, preferably with an interleaved material, such as a fibrous nonwoven material, to prevent layer-to-layer contact of t:he membrane. Most preferably, the membrane: is heat-treated in roll form interleaved with a polyester fibrous nonwoven material.
The heat-treatment can be carried out by any suitable: means. for example, the polyvinylidene fluoride can be subjected t.o the aforesaid temperature by contacting the membrane with a heated surface.
Alternatively, the membrane can be subjected to the desired temperature by placing the membrane, preferably 3o in roll form, in a suitable oven, e.g., a circulating air oven.
The heat-treatment of polyvinylidene fluoride membranes is more fully described in U.S. Patents 5,1,96,508 and 5,198,505. Those patents also describe certain improvements in surface modifications which can be obtained (and are desirably exhibited by the polyvinylidene fluoride membranes in the context of the WO 96132643 PCT/~TS96/04139 present invention) by heat-treating polyvinylidene fluoride membranes, albeit without an appreciation of the surprisingly reduced tendency of detecting reagents to adhere to such membranes in the context of the present invention.
A comparison of polyvinylidene fluoride membranes before and after heat-treatment has led to the discovery that the heat-treatment reduces the surface area of the membrane (as determined by BET analysis). While not seeking' to be bound to any particular theory explaining the surprisingly reduced tendency of detecting reagents to adhere to a heat-treated polyvinylidene fluoride membrane in the context of the present invention, it is believed that the reduced tendency of detecting reagents to adhere to the heat-treated polyvinylidene fluoride membrane is at least in part the result of the reduced surface area of the heat-treated membrane. Detecting reagents apparently have a greater difficulty in adhering to the reduced-surface area polyvinylidene fluoride 2o membrane, while the ability of biological materials, such as proteins, to adhere to the polyvinylidene fluoride membrane is not significantly altered. Thus, the background noise level is decreased, while improving the sensitivity or signal-to-noise ratio of the overall process.
The following examples further illustrate the present invention and, of course, should not be construed as in any way limiting its scope.
Example 1 This example illustrates the superiority of the present inventive method utilizing a heat-treated polyvinylidene fluoride membrane as compared to the same method utilizing a non-heat-treated polyvinylidene fluoride membrane.
A western transfer of a standard test protein mixture from a ge.l to a membrane was carried out using a WO 9f»/32643 PCT/LTS96/~4139 non-heat-treated polyvinylidene fluoride membrane (specif:ically, FlixoroTrans~ from Pail Corporation) and the same membrane which had been subjected in roll form to about x.40 °C in an air circulating oven for about 48 hours (:including t:he heat-up and cool-down time for the oven). The general protein transfer protocol set forth in the product in:~ert for FluoroTransTM was followed, using amido black stain as the detecting reagent. The respectiva membranes were then evaluated with a densitometer.
The' background intensity level (i.e., noise level) for the non-heat-treated polyvinylidene fluoride membrane was nearly twice that for the heat-treated polyvinylidene fluoridsa membrane. Moreover, the ratio of the intensity I5 of the highest peak to the background level was about 1.5:1 far the non-heat-treated .polyvinylidene fluoride membrane and about: 2.8:1 for the heat-treated polyvinylidene fluoride membrane. Thus, the reduction in the bacl~:ground intensity level was accompanied by an improvement in the: signal-to-noise ratio of over 100%.
Example 2 This example further illustrates the superiority of the pre~.ent inventive method utilizing a heat-treated polyvinylidene fluoride membrane as compared to commercially available protein transfer membranes.
Dyed protein markers were electrophoresed and transferred to (a) a non-heat-treated polyvinylidene fluoride: membrane having a pore rating of 0.2 um (specifically, FluoroTransTM from Pall Corporation), (b) a heat-treated polyvinylidene fluoride membrane having a pore rating of 0.2 ~Cm (specifically, FluoroTransTM from Pall Corporation subjected in roll form to about 140 °C
in an ai.r circulating oven for about 48 hours (including the heat.-up and cool-down time for the oven)), (c) a similarly heat-treated polyvinylidene fluoride membrane having a pore rating of 0.45 ~Cm, (d) a commercially-available, competitive polyvinylidene fluoride protein-transfer membrane having a pore rating of 0.45 Vim, and a commerciall~~-available, competitive nitrocellulose membrane: having a pore rating of 0.45 ~cm.
A polyvinylidene fluoride capture membrane was placed behind eactl of the membranes being evaluated so as to capture any protein which passed through the evaluated membranes and to thereby detect any protein "burn through." The protein transfer was allowed to proceed for 24 riours,.and then the capture membranes were examined for the x~resence of proteins.
The non-heat-treated 0.2 ~m pore rated polyvinylidene fluoride membrane (sample a) exhibited no significant protein "burn through." Similarly, the heat-treated 0.2 ~,m pore rated polyvinylidene fluoride membrane, (sample b) exhibited no significant protein "burn through." 'These results demonstrate that the heat-treatment process does not adversely affect the excellent properties of pol~winylidene fluoride membranes of 2o relatively small pore rating as regards protein "burn through ., "
As regards the other membranes which were evaluated, the commercially-available competitive 0.45 ~cm pore rated polyvinylidene fluoride membrane (sample d) exhibited a small, but signif~:cant, amount of protein "burn through,"
while the competitive 0.45 ~m pore rated nitrocellulose membrane (sample ea) exhibited a high degree of protein "burn through. " 7:n contrast, the heat-treated 0 . 45 ~Cm pore rated polyvinylidene fluoride membrane (sample c) exhibited no significant protein "burn through." These results demonstrate that the present inventive method , results in at least an equivalent, if not lower, level of protein "burn through" as compared to methods utilizing similar pore-rated, commercially-available, competitive membranes. As a result, the present inventive method can be expected to re:ault in at least as good, if not better, protein retention,, and ultimately greater sensitivity, thin methods involving those commercially-available, competitive membranes which were evaluated herein.
While this invention has been described with an emphasis upon preferred embodiments, it will be obvious to those of ordinary skill in the art that variations of the preferred products and methods may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims.
Claims (19)
1. A method of detecting a biological material whereby a biological material is adhered to a membrane which is then contacted with a reagent capable of binding to said biological material and capable of being detected, such that the detection of said reagent bound to said biological material represents a biological material detection signal evidencing the presence of said biological material, and the detection of said reagent directly bound to said membrane represents background noise, wherein the improvement comprises utilizing a polyvinylidene fluoride membrane which, prior to adhering said biological material, has been subjected to a temperature of at least about 80 °C but less than the temperature at which the membrane softens and deforms for a time sufficient to substantially reduce said detecting reagent from directly adhering to said membrane, so as to result in a decrease in the background noise and an increase in a biological material detection signal-to-noise ratio.
2. A method of transferring a biological material from one location to a membrane by adhering said biological material to said membrane, wherein the improvement comprises utilizing a polyvinylidene fluoride membrane which, prior to transferring said biological material, has been subjected to a temperature of at least 80°C but less than the temperature at which the membrane softens and deforms for a time sufficient to reduce the ability of another material directly adhere to said membrane as compared to the ability of said another material to directly adhere to said membrane prior to said membrane being subjected to said temperature.
3. The method of claim 1 or 2, wherein said membrane is subjected to a temperature 80°C to 160°C.
4. The method of claim 3, wherein said membrane is subjected to said temperature for 5 minutes to 64 hours.
5. The method of claim 1 or 2, wherein said membrane is subjected to a temperature of 80°C to 150°C.
6. The method of claim 1 or 2, wherein said membrane is subjected to a temperature of 100°C to 150°C.
7. The method of claim 6, wherein said membrane is subjected to said temperature for 32 hours or more.
8. The method of claim 1 or 2, wherein said membrane is subjected to a temperature of 120°C to 150°C.
9. The method of claim 8, wherein said membrane is subjected to said temperature for 16 hours or more.
10. The method of claim 1 or 2, wherein said membrane is subjected to a temperature of 135°C to 145°C.
11. The method of claims 10, wherein said membrane is subjected to said temperature for 12 hours or more.
12. The method of 10, wherein said membrane is subjected to said temperature for 24 hours or more.
13. The method of any one of claims 1 to 12, wherein said membrane is subjected to said temperature by contacting said membrane with a heated surface.
14. The method of any one of claims 1 to 12, wherein said membrane is subjected to said temperature by placing said membrane in a circulating air oven.
15. The method of claim 14, wherein said membrane is in the form of a roll when placed in said circulating air oven.
16. The method of any one of claims 1 to 15, wherein said membrane has a pore rating of 1 µm or less.
17. The method of any one of claims 1 to 15, wherein said membrane has a pore rating of 0.5 µm or less.
18. The method of any one of claims 1 to 15, wherein said membrane has a pore rating of 0.05 µm to 0.45 µm.
19. The method of any one of claims 1 to 15, wherein said membrane has a pore rating of 0.05 µm to 0.2 µm.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/420,218 | 1995-04-11 | ||
| US08/420,218 US5567626A (en) | 1995-04-11 | 1995-04-11 | Method of detecting biological materials using a polyvinyllidene fluoride membrane |
| PCT/US1996/004139 WO1996032643A1 (en) | 1995-04-11 | 1996-03-20 | Method of detecting biological materials using a polyvinylidene fluoride membrane |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2212193A1 CA2212193A1 (en) | 1996-10-17 |
| CA2212193C true CA2212193C (en) | 2001-10-09 |
Family
ID=23665554
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002212193A Expired - Lifetime CA2212193C (en) | 1995-04-11 | 1996-03-20 | Method of detecting biological materials using a polyvinylidene fluoride membrane |
Country Status (7)
| Country | Link |
|---|---|
| US (1) | US5567626A (en) |
| EP (1) | EP0820592B1 (en) |
| JP (1) | JP2996514B2 (en) |
| CA (1) | CA2212193C (en) |
| DE (1) | DE69603716T2 (en) |
| GB (1) | GB2313836B (en) |
| WO (1) | WO1996032643A1 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050173341A1 (en) * | 2002-05-03 | 2005-08-11 | Pall Corporation | Blended polymer media for treating aqueous fluids |
| US20080073622A1 (en) * | 2006-09-22 | 2008-03-27 | Inolex Investment Corporation | Conductive polyurethane foam and methods for making same |
| JP2009249439A (en) * | 2008-04-02 | 2009-10-29 | Lintec Corp | Modified polyvinylidene fluoride membrane, laminated membrane for protein adsorption, and manufacturing method thereof |
| JP2011242159A (en) * | 2010-05-14 | 2011-12-01 | Yokohama City Univ | Method for transferring protein separated by electrophoresis onto membrane filter and performing mass spectrometry |
Family Cites Families (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4302204A (en) * | 1979-07-02 | 1981-11-24 | The Board Of Trustees Of Leland Stanford Junior University | Transfer and detection of nucleic acids |
| US4341605A (en) * | 1981-01-16 | 1982-07-27 | E. I. Du Pont De Nemours And Company | Process for cation permeable membrane with reinforcement fabric embedded therein and product thereof |
| US4455370A (en) * | 1982-05-17 | 1984-06-19 | E. I. Du Pont De Nemours And Company | Transferring separated components in gel electrophoresis via nylon membrane |
| US4618533A (en) * | 1984-11-30 | 1986-10-21 | Millipore Corporation | Porous membrane having hydrophilic surface and process |
| US4923620A (en) * | 1987-10-20 | 1990-05-08 | Pall Corporation | Device for depletion of the leukocyte content of blood and blood components |
| US5004543A (en) * | 1988-06-21 | 1991-04-02 | Millipore Corporation | Charge-modified hydrophobic membrane materials and method for making the same |
| US5200312A (en) * | 1989-01-30 | 1993-04-06 | The United States Of America As Represented By The Secretary Of The Navy | Membrane based dot immunoassay and method of use |
| US5234809A (en) * | 1989-03-23 | 1993-08-10 | Akzo N.V. | Process for isolating nucleic acid |
| US5071909A (en) * | 1989-07-26 | 1991-12-10 | Millipore Corporation | Immobilization of proteins and peptides on insoluble supports |
| US5079272A (en) * | 1989-11-30 | 1992-01-07 | Millipore Corporation | Porous membrane formed from interpenetrating polymer network having hydrophilic surface |
| US5198505A (en) * | 1991-04-11 | 1993-03-30 | Pall Corporation | Uniform polyvinylidene difluoride membranes |
| US5196508A (en) * | 1991-04-11 | 1993-03-23 | Pall Corporation | Method for making uniform polyvinylidene difluoride membranes |
| CA2071736A1 (en) * | 1991-06-24 | 1992-12-25 | Naoyuki Kouno | Acid dye staining method |
| US5240615A (en) * | 1991-08-20 | 1993-08-31 | Fishman Jerry H | Composite membrane composed of microporous polyvinylidene difluoride membrane laminated to porous support and process for its preparation |
| US5183607A (en) * | 1991-12-17 | 1993-02-02 | Beall George H | Polymer membranes for separation process |
| US5283186A (en) * | 1991-12-31 | 1994-02-01 | Abbott Laboratories | Preparation of a compressed membrane containing immobilized biologically acting material |
-
1995
- 1995-04-11 US US08/420,218 patent/US5567626A/en not_active Expired - Lifetime
-
1996
- 1996-03-20 WO PCT/US1996/004139 patent/WO1996032643A1/en active IP Right Grant
- 1996-03-20 EP EP96909874A patent/EP0820592B1/en not_active Expired - Lifetime
- 1996-03-20 JP JP8531024A patent/JP2996514B2/en not_active Expired - Lifetime
- 1996-03-20 GB GB9716671A patent/GB2313836B/en not_active Expired - Lifetime
- 1996-03-20 CA CA002212193A patent/CA2212193C/en not_active Expired - Lifetime
- 1996-03-20 DE DE69603716T patent/DE69603716T2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH10508106A (en) | 1998-08-04 |
| US5567626A (en) | 1996-10-22 |
| DE69603716D1 (en) | 1999-09-16 |
| GB2313836A (en) | 1997-12-10 |
| WO1996032643A1 (en) | 1996-10-17 |
| DE69603716T2 (en) | 2000-01-13 |
| GB9716671D0 (en) | 1997-10-15 |
| JP2996514B2 (en) | 2000-01-11 |
| EP0820592A1 (en) | 1998-01-28 |
| EP0820592B1 (en) | 1999-08-11 |
| GB2313836B (en) | 1999-05-05 |
| CA2212193A1 (en) | 1996-10-17 |
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| EEER | Examination request | ||
| MKEX | Expiry |
Effective date: 20160321 |