WO1996032643A1 - Method of detecting biological materials using a polyvinylidene fluoride membrane - Google Patents

Method of detecting biological materials using a polyvinylidene fluoride membrane Download PDF

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Publication number
WO1996032643A1
WO1996032643A1 PCT/US1996/004139 US9604139W WO9632643A1 WO 1996032643 A1 WO1996032643 A1 WO 1996032643A1 US 9604139 W US9604139 W US 9604139W WO 9632643 A1 WO9632643 A1 WO 9632643A1
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Prior art keywords
membrane
temperature
subjected
polyvinylidene fluoride
hours
Prior art date
Application number
PCT/US1996/004139
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French (fr)
Inventor
Richard Frank Salinaro
Original Assignee
Pall Corporation
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Pall Corporation filed Critical Pall Corporation
Priority to JP8531024A priority Critical patent/JP2996514B2/en
Priority to GB9716671A priority patent/GB2313836B/en
Priority to DE69603716T priority patent/DE69603716T2/en
Priority to CA002212193A priority patent/CA2212193C/en
Priority to EP96909874A priority patent/EP0820592B1/en
Publication of WO1996032643A1 publication Critical patent/WO1996032643A1/en

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Classifications

    • 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
    • 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

Definitions

  • the present invention 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.
  • 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) .
  • biological material of interest adheres to 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.
  • a detecting reagent e.g., a dye or stain
  • 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.
  • 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.
  • the present invention involves the use of a heat- treated polyvinylidene fluoride membrane in a method of detecting or transferring a biological material.
  • the present invention provides for the use of a heat-treated polyvinylidene fluoride membrane in a method of detecting or transferring a biological material whereby a biological material is adhered to the membrane which is then contacted with a detecting reagent.
  • 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.
  • 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
  • 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 polyvinylidene 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 ⁇ m or less, more typically about 1 ⁇ m or less, e.g., about 0.01-1 ⁇ m, and most typically about 0.50 ⁇ m or less.
  • the membrane has a pore rating of about 0.05-0.45 ⁇ m or, even more preferably, about 0.05-0.2 ⁇ m.
  • the polyvinylidene fluoride membrane can be heat- treated, or annealed, in any suitable manner, desirably at a temperature and for a period of time sufficient to reduce the ability of the detecting reagent to directly adhere to the membrane.
  • the heat- treatment preferably results in a polyvinylidene fluoride membrane to which the detecting reagent is less likely to adhere as compared to the non-heat-treated polyvinylidene fluoride membrane.
  • the 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.
  • 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.
  • the upper temperature limit will be about 160 °C. The amount of time of heating will vary with the heating temperature and nature of the membrane being heated.
  • 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.
  • the membrane is preferably subjected to about 160 °C for about 5 minutes or more, more preferably about 10 minutes or more.
  • 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, for a suitable period of time, desirably about 32 hours or more. More preferably, the membrane is subjected to a temperature of about 120 °C to about 150 ⁇ C for a suitable period of time, desirably about 16 hours or more, e.g., about 48-72 hours.
  • the membrane is subjected to a temperature of about 135 °C to about 145 °C, 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 effected 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., shrinkage. Any suitable means can be used to dimensionally restrain the membrane.
  • the membrane can be placed 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 the membrane.
  • 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.
  • the polyvinylidene fluoride can be subjected to the aforesaid temperature by contacting the membrane with a heated surface.
  • the membrane can be subjected to the desired temperature by placing the membrane, preferably in roll form, in a suitable oven, e.g., a circulating air oven.
  • Detecting reagents apparently have a greater difficulty in adhering to the reduced-surface area polyvinylidene fluoride membrane, while the ability of biological materials, such as proteins, to adhere to the polyvinylidene fluoride membrane is not significantly altered.
  • the background noise level is decreased, while improving the sensitivity or signal-to-noise ratio of the overall process.
  • 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 gel to a membrane was carried out using a non-heat-treated polyvinylidene fluoride membrane (specifically, FluoroTransTM from Pall Corporation) and the same membrane which had been subjected in roll form to about 140 °C in an air circulating oven for about 48 hours (including the heat-up and cool-down time for the oven) .
  • the general protein transfer protocol set forth in the product insert for FluoroTransTM was followed, using amido black stain as the detecting reagent. The respective 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 fluoride membrane. Moreover, the ratio of the intensity of the highest peak to the background level was about 1.5:1 for the non-heat-treated polyvinylidene fluoride membrane and about 2.8:1 for the heat-treated polyvinylidene fluoride membrane. Thus, the reduction in the background 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 present 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 ⁇ m (specifically, FluoroTransTM from Pall Corporation) , (b) a heat-treated polyvinylidene fluoride membrane having a pore rating of 0.2 ⁇ m (specifically, FluoroTransTM from Pall Corporation subjected in roll form to about 140 °C in an air 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 ⁇ m, (d) a commercially- available, competitive polyvinylidene fluoride protein- transfer membrane having a pore rating of 0.45
  • a polyvinylidene fluoride capture membrane was placed behind each 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 hours, and then the capture membranes were examined for the presence of proteins.
  • the commercially-available competitive 0.45 ⁇ m pore rated polyvinylidene fluoride membrane (sample d) exhibited a small, but significant, amount of protein "burn through,” while the competitive 0.45 ⁇ m pore rated nitrocellulose membrane (sample e) exhibited a high degree of protein "burn through.”
  • the heat-treated 0.45 ⁇ m pore rated polyvinylidene fluoride membrane (sample c) exhibited no significant protein "burn through.”

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.

Description

METHOD OF DETECTING BIOLOGICAL MATERIALS USING A POLYVINYLIDENE FLUORIDE MEMBRANE
TECHNICAL FIELD OF THE INVENTION The present invention 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.
BACKGROUND 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 to 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 provides such an improvement through the use of a modified polyvinylidene fluoride membrane which exhibits reduced background noise. These and other objects and advantages of the present invention, as well as additional inventive 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 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.
DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention involves the use of a heat- treated polyvinylidene fluoride membrane in a method of detecting or transferring a biological material. In particular, the present invention provides for the use of a heat-treated polyvinylidene fluoride membrane in a method of detecting or transferring a biological material whereby a biological material is adhered to the membrane which is then contacted 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. 7 , 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.. 9£, 503-517 (1975). These detection or transfer methods, as well as numerous variations thereon and other detection or transfer procedures utilizing membranes, particularly hydrophobie 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 FluoroTrans™ 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 polyvinylidene 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 μm or less, more typically about 1 μm or less, e.g., about 0.01-1 μm, and most typically about 0.50 μm or less. Preferably, the membrane has a pore rating of about 0.05-0.45 μm or, even more preferably, about 0.05-0.2 μm. The polyvinylidene fluoride membrane can be heat- treated, or annealed, in any suitable manner, desirably at a temperature and for a period of time sufficient to reduce the ability of the detecting reagent to directly adhere to the membrane. In other words, the heat- treatment preferably results in a polyvinylidene fluoride membrane to which the detecting reagent is less likely to adhere as compared to the non-heat-treated polyvinylidene fluoride membrane.
Preferably, the 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 heating 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., less than one minute, to heat, while a rolled membrane of several hundred linear meters may require many hours 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, for a suitable period of time, desirably about 32 hours or more. More preferably, the membrane is subjected to a temperature of about 120 °C to about 150 βC for a suitable period of time, desirably about 16 hours or more, e.g., about 48-72 hours. Most preferably, the membrane is subjected to a temperature of about 135 °C to about 145 °C, 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 effected 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., shrinkage. Any suitable means can be used to dimensionally restrain the membrane. For example, the membrane can be placed 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 the 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 to 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 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 5,196,508 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 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 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 gel to a membrane was carried out using a non-heat-treated polyvinylidene fluoride membrane (specifically, FluoroTrans™ from Pall Corporation) and the same membrane which had been subjected in roll form to about 140 °C in an air circulating oven for about 48 hours (including the heat-up and cool-down time for the oven) . The general protein transfer protocol set forth in the product insert for FluoroTrans™ was followed, using amido black stain as the detecting reagent. The respective 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 fluoride membrane. Moreover, the ratio of the intensity of the highest peak to the background level was about 1.5:1 for the non-heat-treated polyvinylidene fluoride membrane and about 2.8:1 for the heat-treated polyvinylidene fluoride membrane. Thus, the reduction in the background 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 present 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 μm (specifically, FluoroTrans™ from Pall Corporation) , (b) a heat-treated polyvinylidene fluoride membrane having a pore rating of 0.2 μm (specifically, FluoroTrans™ from Pall Corporation subjected in roll form to about 140 °C in an air 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 μm, (d) a commercially- available, competitive polyvinylidene fluoride protein- transfer membrane having a pore rating of 0.45 μm, and (e) a commercially-available, competitive nitrocellulose membrane having a pore rating of 0.45 μm. A polyvinylidene fluoride capture membrane was placed behind each 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 hours, and then the capture membranes were examined for the presence of proteins.
The non-heat-treated 0.2 μm pore rated polyvinylidene fluoride membrane (sample a) exhibited no significant protein "burn through." Similarly, the heat- treated 0.2 μm pore rated polyvinylidene fluoride membranes (sample b) exhibited no significant protein "burn through." These results demonstrate that the heat- treatment process does not adversely affect the excellent properties of polyvinylidene fluoride membranes of relatively small pore rating as regards protein "burn through."
As regards the other membranes which were evaluated, the commercially-available competitive 0.45 μm pore rated polyvinylidene fluoride membrane (sample d) exhibited a small, but significant, amount of protein "burn through," while the competitive 0.45 μm pore rated nitrocellulose membrane (sample e) exhibited a high degree of protein "burn through." In contrast, the heat-treated 0.45 μm 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 result in at least as good, if not better, protein retention, and ultimately greater sensitivity, than methods involving those commercially-available, competitive membranes which were evaluated herein.
All of the references cited herein are hereby incorporated in their entireties by reference.
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

WHAT IS CLAIMED IS:
1. A method of detecting a biological material whereby a biological material is adhered to a membrane which is then contacted with a detecting reagent to detect the biological material adhered to said membrane, 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 said detecting reagent to directly adhere to said membrane as compared to the ability of said detecting reagent to directly adhere to said membrane prior to said membrane being subjected to said temperature.
2. The method of claim 1, wherein said membrane is subjected to a temperature of about 80 °C to about 160 °C.
3. The method of claim 2, wherein said membrane is subjected to said temperature for about 5 minutes to about 64 hours.
4. The method of claim 1, wherein said membrane is subjected to a temperature of about 80 °C to about 150 °C.
5. The method of claim 1, wherein said membrane is subjected to a temperature of about 100 °C to about 150
°C.
6. The method of claim 5, wherein said membrane is subjected to said temperature for about 32 hours or more.
7. The method of claim 1, wherein said membrane is subjected to a temperature of about 120 °C to about 150 °C.
8. The method of claim 7, wherein said membrane is subjected to said temperature for about 16 hours or more.
9. The method of claim 1, wherein said membrane is subjected to a temperature of about 135 °C to about 145 °C.
10. The method of claim 9, wherein said membrane is subjected to said temperature for about 12 hours or more.
11. The method of claim 10, wherein said membrane is subjected to said temperature for about 24 hours or more.
12. The method of any of claims 1-11, wherein said membrane is subjected to said temperature by contacting said membrane with a heated surface.
13. The method of any of claims 1-11, wherein said membrane is subjected to said temperature by placing said membrane in a circulating air oven.
14. The method of claim 13, wherein said membrane is in the form of a roll when placed in said circulating air oven.
15. The method of any of claims 1-14, wherein said membrane has a pore rating of about 1 μm or less.
16. The method of claim 15, wherein said membrane has a pore rating of about 0.5 μm or less.
17. The method of claim 16, wherein said membrane has a pore rating of about 0.05-0.45 μm.
18. The method of claim 17, wherein said membrane has a pore rating of about 0.05-0.2 μm.
19. A method of transferring a biological material with a membrane, 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 another material which is not said biological material to directly adhere to said membrane as compared to the ability of said other material to directly adhere to said membrane prior to said membrane being subjected to said temperature.
20. The method of claim 19, wherein said membrane is subjected to a temperature of about 80 °C to about 160
°C.
21. The method of claim 20, wherein said membrane is subjected to said temperature for about 5 minutes to about 64 hours.
22. The method of claim 19, wherein said membrane is subjected to a temperature of about 80 °C to about 150 °C.
23. The method of claim 19, wherein said membrane is subjected to a temperature of about 100 °C to about 150 °C.
24. The method of claim 23, wherein said membrane is subjected to said temperature for about 32 hours or more.
25. The method of claim 19, wherein said membrane is subjected to a temperature of about 120 °C to about 150 βC.
26. The method of claim 25, wherein said membrane is subjected to said temperature for about 16 hours or more.
27. The method of claim 19, wherein said membrane is subjected to a temperature of about 135 °C to about
145 °C.
28. The method of claim 27, wherein said membrane is subjected to said temperature for about 12 hours or more.
29. The method of claim 28, wherein said membrane is subjected to said temperature for about 24 hours or more.
30. The method of any of claims 19-29, wherein said membrane is subjected to said temperature by contacting said membrane with a heated surface.
31. The method of any of claims 19-29, wherein said membrane is subjected to said temperature by placing said membrane in a circulating air oven.
32. The method of claim 31, wherein said membrane is in the form of a roll when placed in said circulating air oven.
33. The method of any of claims 19-32, wherein said membrane has a pore rating of about 1 μm or less.
34. The method of claim 33, wherein said membrane has a pore rating of about 0.5 μm or less.
35. The method of claim 34, wherein said membrane has a pore rating of about 0.05-0.45 μm.
36. The method of claim 35, wherein said membrane has a pore rating of about 0.05-0.2 μm.
PCT/US1996/004139 1995-04-11 1996-03-20 Method of detecting biological materials using a polyvinylidene fluoride membrane WO1996032643A1 (en)

Priority Applications (5)

Application Number Priority Date Filing Date Title
JP8531024A JP2996514B2 (en) 1995-04-11 1996-03-20 Method for detecting biological material using polyvinylidene fluoride membrane
GB9716671A GB2313836B (en) 1995-04-11 1996-03-20 Method of detecting biological materials using a polyvinylidene fluoride membrane
DE69603716T DE69603716T2 (en) 1995-04-11 1996-03-20 METHOD FOR FINDING BIOLOGICAL SUBSTANCES BY MEANS OF A POLYVINYLIDENE FLUORIDE MEMBRANE.
CA002212193A CA2212193C (en) 1995-04-11 1996-03-20 Method of detecting biological materials using a polyvinylidene fluoride membrane
EP96909874A EP0820592B1 (en) 1995-04-11 1996-03-20 Method of detecting biological materials using a polyvinylidene fluoride membrane

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US08/420,218 US5567626A (en) 1995-04-11 1995-04-11 Method of detecting biological materials using a polyvinyllidene fluoride membrane
US08/420,218 1995-04-11

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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

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EP0820592A1 (en) 1998-01-28
JP2996514B2 (en) 2000-01-11
GB2313836B (en) 1999-05-05
GB9716671D0 (en) 1997-10-15
US5567626A (en) 1996-10-22
CA2212193C (en) 2001-10-09
CA2212193A1 (en) 1996-10-17
GB2313836A (en) 1997-12-10
DE69603716D1 (en) 1999-09-16
DE69603716T2 (en) 2000-01-13
EP0820592B1 (en) 1999-08-11

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