JPH0643152A - Solid-phase assay system solidifying high molecular body in solid phase - Google Patents

Abstract

PURPOSE: To obtain an excellent solid phase assay system for solidifying a polymer, e.g. nucleic acid or protein, into a solid phase from a chromatograph substrate, having a fixed matrix on the surface thereof by employing a fixed matrix having high adsorption capacity and good preservation properties for solidifying a polymer more efficiently as the solid phase. CONSTITUTION: A cation charge modified microporous nylon film, i.e., a hydrophilic microporous nylon film having pores over the entire film, and the surface of the pore modified with a cation charge modifier is employed as a fixed matrix.

Description

Detailed Description of the Invention [0001] FIELD OF THE INVENTION The present invention relates to a charge-modified microporous material.
Blotting of macromolecules using membranes, especially nucleic acids and
Regarding protein blotting. For more details,
Ming is a cationic matrix on its surface as a fixed matrix.
Chromatograph with load-modified microporous nylon membrane
Solid-phase assay for immobilizing polymer from solid substrate in solid phase
Regarding the system. [0002] [Prior Art] "Smithies, Zone Electrophoresis in
StarchGels: Group Variations in the Serum Proteins
of Normal Human Adults ”[Biochem. J. 61: 629641
(1955)], starch gel as a molecular sieve
A note that is useful and through which zone electrophoresis occurs.
Can be seen. Since then, gel electrophoresis technology has always been reliable
Has made great progress. Acrylamide gel or discontinuous buffer
Introducing the system, unfolding protein complex resolved on gel
Use of sodium dodecyl sulfate (SDS) for cormorant
Buffer for polyacrylamide gel electrophoresis
The combination of SDS in the system is a part of modern biology.
Have made a significant contribution to the development of analysis and preparation tools.
It was The main purpose of these techniques is to give a given experimental treatment.
Special "bands" may appear throughout the period of the placement.
Or the disappearance of the protein sample
To be able to see the complexity. One-dimensional gel
Is a virus, bacteriophage, erythrocyte ghostmen
Analyze only relatively simple protein samples such as Bran
It turns out to be suitable. In a more complex system
Is a new two-dimensional gel because a greater resolution was required.
The system was developed. Today, the most complex proteinaceous tests
Even the myriad of polypeptides, one of the
It came to be resolved. Known one "band" or "spot"
The task of correctly correlating with the function of is often difficult
And this means that the resolution of proteins is based on protein denaturation.
It is especially difficult if Nevertheless,
Specific enzyme or antigen or glycoprotein or formo in gel
Many means have been developed to allow identification of the receptor.
It was These methods maintain at least one of the following prerequisites:
Depends on ability to have: (1) polypeptide
Remain active during electrophoresis; (2) denatured po
Removal of denaturation of lipid peptide; and (3) Detection during electrophoresis
Search for possible ligands Covalent cross-linking of proteins. So
Besides, many steps and long-term
With incubation and washing steps. This is time
It is a waste of time, and the gel breaks, tears or dries.
Tendency to cause accidents like gel cracking during the period
Is often seen. Overcoming problems in gel analysis
To this end, new methods have been developed. That is, from an agarose gel to D
To transfer the NA pattern to the nitrocellulose membrane
“Southern-blotting”, an established method of
The method also applies to protein patterns on polyacrylamide gels
Published by a wide variety of literature that revealed that
Has been done. Raw protein pattern elutes from the gel
And is immobilized on the base layer. On the other hand, the base layer is a "band"
Or suitable for gels to identify “spots”
You can perform the same operations that have been used. However,
Moving the cutrophorogram to a fixed matrix
Should yield the following benefits: 1) Moisture-fixing matrix is flexible and easy to handle; 2) The immobilized protein easily and evenly approaches various ligands.
Sui (In the gel by Differential Polarity
It is due to the various restrictions introduced are lifted); 3) Generally, migration analysis can be performed on small samples; 4) processing
The time (incubation and washing) can be significantly reduced
R; 5) Many replicas can be made; 6) Transfer pattern can be stored for several months before use; 7) This protein transfer can tolerate multiple analyses.
Moreover, this transferred protein pattern is usually
Analysis methods that are extremely difficult or impossible to
Easy to accept. At present, the term "blotting"
Fixing biological macromolecules such as nucleic acids and proteins from gels
A way to get to Trix. This expression is
The name of the macromolecule involved in
DNA blotting and RNA blotting
Used like a ring. Raw containing immobilized transfer protein
The growth matrix is either "blot" or "transfer"
-"And this is an ink depending on the ligand.
It can be incubated. This operation is called "overlay"
It is one method called. For example, immune-overlay
(Immuno-overlay), lectin overlay or carmo
Dallin (calmodulin) overlays are antibodies,
Incubation of blots with lectin or calmodarin
Refers to the solution. DN which is a kind of nucleic acid blotting
A-blotting is performed by [J. Mol. Biol. 98: 503-517 (197
5)] to “Detection of Specific Sequences among DNA
 Fragment Separated by Gel Erectrophoresis "
Called "Southern Transfer" written by Southern et al.
It is based on the technology used. Chromatographic separation of DNA fragments
After this, the DNA is denatured in a gel and then the gel
Neutralize the battery. Contact the gel with a buffer reservoir
Gauze heart paper
Place on top of the gel, dry blotting paper
Is provided on top of this nitrocellulose. Passing through the gel
A stream of material that elutes the DNA, which in turn
It binds to rocellulose. Thus, electrophoretically separated
The DNA fragment pattern is nitrocellulose
It is moved to the top and saved. Use specific labeled nucleic acid
And cross-breeding, the complementary floes bound to nitrocellulose.
It can detect the lagment. Single-stranded RNA and DNA are
Cellulose powder substituted with aminobenzyloxymethyl group
Diazotization of the amine of diazobenzylmethyl (DB
M) -Cellulose powder activated by forming cellulose
It was discovered in 1976 that it could be covalently bound to. DNA
Is difficult to combine with nitrocellulose,
This made it difficult or impossible to apply the r "method.
The facts of the facts have filled the gap of the crossbreeding technology. 19
In 1977,Proc.Natl.Acad.Sci.US A.74: 53
50-5354 (1977)] in “Method for Detection
of Specific RNAs in Agarose Gels by Transfer to Di
azobenzyloxymethyl-Paper and Hybridization whih DN
A paper entitled "A Probes" was published by Alwine et al.
Here, the following structural formula 3 [Chemical 3] Diazobenzyloki called DBM-paper shown in
Cellulosic / fibrous sheet modified with dimethyl group
That is, a blotting paper) was made. This D
BM-paper is an electrophoretically separated RNA pattern
From agarose gel similar to “Souther Transfer” method
It was announced that it could be used to move in any way. Diazo
Type (DPT-paper) activated aminophenylthio
Oether paper is used as well. Both
DNA, RNA and protein covalently and irreversibly
Combine with. DBM-paper and DPT-paper are
It needs activation and has a limited life, so
It is unstable and its binding capacity is simply that of nitrocellulose.
Comparable and irreversibly bound to polymer
The drawback is that it hinders elution in the next step,
And the problem that resolution is difficult due to the surface structure
Have a point. Even a little inconvenience of DBM paper
Efforts to submit were made by Thomas (1980), but high
RNA and small amounts of DNA fragments using salinity
A technique has been developed to transfer corn to nitrocellulose. R
NA binding efficiency is 35 ug / cm for DBM paper² compared to
80 ug / cm² I found out that Polyacrylamide
Elution of macromolecules from gels is effective by electrophoresis
I can carry it out. However, high current is required because high salt concentration is required.
Requires and is not practical. First developed in the context of DNA and RNA research
The Southern blotting technology that was issued
It has become recognized that it may be applicable
It was This protein transfer technology was first developed by Renart.
hand,〔Proc.Natl.Acad.Sci.USA.76: 3116 ~ 3120
(1979)] "Transfer of Proteins from Gels to Di
azobenzyloxymethyl-Paper and Detection With Antise
"Ra" was announced. Renart is a composite agarose-a
Carry out transfer of protein to DBM using Crylamide gel
However, the acrylamide cross-linking here is reversible.
It was After electrophoresis, the cross-links are released and the low%
Separate the agarose gel and easily transfer the protein from this gel.
Moved. Immediately after this, Bowen et al.
To develop theNuc.Acids.Res.8: 1-20 (1980)]
`` The Detection of DNA-Binding Proteins by Protein
 Blotting "was announced. Here, SDS-Po
DNA-binding proteins separated on a polyacrylamide gel
And nitrocellulose for separation of other ligand-binding proteins
Was used. The movement was by diffusion. Towbin et al.Pr
oc.Natl.Acad.Sci.USA 76: 4350-4354 (1979)]
By the way, Bittner et al.Anal.Biochem.102: 459
~ 471 (1980)], even at low salinity
It was shown that migration was possible electrophoretically. In general, protein blotting is performed in two steps.
The process consists of the so-called gel elution of the polypeptide
And adsorption of the eluent on a fixed matrix. High molecular
Three types of activation force for body elution were pioneered. One is expansion
It's a day. Here, the gel containing the polymer to be transferred is used.
Pin between two sheets forming a fixed matrix,
This fixed matrix is on the one hand a foam pad and stainless steel
Sandwich between steel screens. Then this combination 2
Submerge in 1 of buffer and leave for 36-48 hours. This a
As a result of the incubation, two identical replica boots
Lot was obtained. The transfer efficiency reached 75%, but this value
Must be split between the two replicas. Matrice
The amount of polymer adsorbed on the chamber is sufficient for the required test.
Yes, and if long travel times are acceptable, due to diffusion
Blotting is useful. The second method of polymer blotting is
Performing a DNA blot using the old method described by Southern
Mass flow of liquid flowing through the gel as well as
Is based on. Place the gel in the buffer reservoir. Matrice
Place a crease on the gel and stack a paper towel on the matrix.
Overlap. This towel is stored through gel and matrix
Absorb buffer from the bath. The movement of the fluid causes the protein to
The protein then acts as a motive force to elute from the
Trapped during This method is faster than the diffusion method
In addition, the elution efficiency is good. Modification of this method
A law has been proposed where bidirectional blockin
I can do it. Besides, by adopting the decompression method
The elution time was dramatically reduced. Most widely used in protein blotting
The method is based on electrophoresing proteins from the gel
There is. This is the opposite of DNA
Adsorbs nitrocellulose even in strong buffers
It is possible due to the fact that Therefore, it is not practical
Proteins from gels without the use of high currents
be able to. The concept of electrophoresis in blotting is
〔Cell.11: 363-370 (1977)] in "Heterogeneity of
 The Ribosomal Genes in Mice and Men "
There is. Many device designs have been reported today,
Rarely are commercially available. The gist is a wet matrix
Place the cus on the gel, at this time air bubbles will also filter
Check that it is not carried between the matrix and gel.
To do. Then, use the matrix and gel with "Scotch Brite"
Cleaning pad, foam rubber or wet blotting page
It is sandwiched between par-like porous support pads. Then
The assembly is supported by a solid grid, usually non-conductive
It The gel and matrix are strongly held to each other
Is very important. Thus establishing a good move
And prevent "distortion" of the protein band. Supported "gel + matrix sandwich"
Insert the "H" into the tank containing the buffer solution, and
Put. The two electrodes that are tacked to the tank wall are
To create a uniform electric field over the entire area of the gel to be
To be installed. Use a conductive continuous sheet for the electrodes.
And theoretically suitable for the purpose. Grapha
Ito slabs and stainless steel plates can also be used. Shi
Operating unit with caulking electrodes has significantly higher current
Is usually impractical because of the need for. Extremely good
A rational, economical and efficient design isAna.Bioche
m.102: 459-471] in the "Electrophoretic Transfer of
 Proteins and Nucleic Acids from SlabGels to Diazo
benzyl oxymethyl Cellulose or Nitrocellulose Sheet
"s". Affects field uniformity
Factors include the distance between the gel and the electrode, the density of the electrode material, and
The distance between each stretch of wire used. In general
The higher the amount of electrode material present, the higher the electrical resistance of the system.
descend. Therefore, it is necessary to drive the elution process
High current is required to obtain a reasonable voltage difference. The transfer buffer used has a low ionic strength.
Such as phosphate buffer, Tris-borate buffer
Or containing methanol, such as tris-glycine
Is used as a non-containing buffer solution. Methanol vs. protein
Tends to increase the binding capacity of nitrocellulose.
And stabilizes the geometry of the gel being transferred, but S
The efficiency of protein elution from DS gel is reduced,
To move efficiently, elute for a long time, generally at 12:00
It has to be done over a period of time. Methanol
If not present, the acrylamide gel will tend to swell.
There is a tendency, and if it is left as it is during protein transfer, it causes cancer
A band arises. The transfer conditions themselves are gel type, fixed matrix
And the moving device used and the type of polymer.
There is a possibility. Native gel, SDS gel, lithi containing gel
Umdodecyl sulphate, iso-electrofocus
2D gels and iso-electro focusing 2D gels
Roth gels are all used for protein blotting
There is. Determine the charge of the eluted protein and load the matrix
Must be placed on the appropriate side of the gel. For example, SDS-
The protein from the gel elutes as anions,
Tricks should be placed on the anode side of the gel. Unchanged
The opposite is true for a sex gel. As electroelution proceeds
As a result, the electrolyte from the gel is also eluted, which buffers it.
A decrease in resistance occurs as a result of contributing to the conductivity of the liquid. Move
If a constant voltage is applied, the current will change accordingly.
Increased to over 1 amp and actually 5 amps
It may reach. Supply of high current such as 1 to 5 amps
Salary is now commercially available in electroblotting
Noh. Another convenient way is a 12V battery charger
Is used, which is considered reasonable. Only
And most of the usual power supplies used for gel electrophoresis
The device cannot exceed 200-250 mA,
Therefore, operating with a maximum constant current such as 200 mA and moving
It was found to be advantageous to gradually reduce the voltage
It was. Low or intermediate molecular weight separated proteins are
May not elute efficiently from the solvent. These proteins
Coincidentally on the isoelectric point, it does not tend to move into the electric field.
That is the case. In such cases, other buffer conditions
Can be adopted. Many fixed matrices are now available
it can. Nitrocellulose is the widest matrix
A commonly used material, but with nitrocellulose and protein
Interactions are complex and not fully understood. For example
Normally, at pH 8 for electroelution of proteins, nitrocellulosics
As the protein is adsorbed, it becomes negatively charged. This interaction
The hydrophilic effect plays a role in the
The elution of proteins from the membrane is affected by the nonionic surfactant.
Will be performed. Some proteins, especially low molecular weight proteins,
Weak binding with trocellulose, during transfer or between next steps
May be lost in. To prevent such loss
To cross-link the transfer polypeptide to the matrix
The protein pattern by covalent bonds.
It is possible to standardize. Nitrocellulose membrane matri
The presence of cellulose acetate in the
The ability seems to decrease. However,
The Cate Matrix was successfully embedded for protein blotting
Is used for. The disadvantages of the nitrocellulose matrix are
Other fixed matrices proposed to improve even in dry
It has come. Transfer to DBM paper is stable with covalent bond
Yields a defined protein pattern. Book compared to membrane matrix
The resolution on the material is slightly lower due to its qualitatively grainy appearance.
Glycine, which is commonly used as a migration buffer, is also available in DBM
Interfere with per protein blotting. So nitrocellulo
Source, DBM paper and other matrices
No, the drawbacks of fixed matrices used in the past
There is still a need to develop an improved fixed matrix
ing. Hope to emerge even with greater mobility
It is rare. Various membranes can be used for chromatography and special
It can be used for electrophoresis technology as a special case
Is known. These are described in the following documents.
Be done. U.S. Pat.No. 3,808,118 No. 3,829,370 No. 3,945,926 publication No. 3,957,651 Publication No. 3,989,613 No. 4,043,895 Publication No. 4,111,784 No. 4,158,683 No. 4,204,929 No. 4,243,507 No. 4,310,408 Japanese Patent No. 4,311,574 Further, the polyamide powder is a chromatographic component.
It is also known that it can be used for separation. U.S. Pat.No. 3,418,158 No. 3,523,350 publication U.S. Pat.No. 4,128,470 discloses nylon micropo
The lath membranes through which the chromatography is performed
Electrophoresis and isoelectric (such as
For isoelectric focusing
It is disclosed that this can be done. New Engl and Nucle
A series of electrophoretic materials called "Gene Screen" by ar.
Uncharged nylon used for blotting as one of the materials
The membrane is on sale. This material is about the binding capacity of proteins
Looks comparable to nitrocellulose. Utility Polymeric blots or transfers should be in a gel
In contrast to what has been developed for various
Of the filter matrix.
The operation does not take time and is inexpensive from the viewpoint of reagents used.
And there are few accidents. More importantly, gel to mat
Diffusion barriers are effectively eliminated when proteins transfer to the lix
Is to be done. Besides, these denatured proteins are
Denaturation is released when SDS is separated from
Often, so the method is probably more convenient
Seems to be effective. Most of the probes currently used are
Polymers, which are especially
It binds well to the constant domain of tide. Lectin is a glycoprotein
And their corresponding antibodies.
It is used to check the connection. Whatever the purpose of the test
, The matrix void without protein band is over
Adversely adsorbs the probe during the laying process, which is harmful
Cause a bad background. So Matrick
The unbound position of the
You have to stand and quench. Normal,
The quenching was carried out at a high concentration of bovine serum albumin (BS
A) or the blot in hemoglobin at 25-60 ° C for 1-
Carried out by incubating for 12 hours
ing. Ovalbumin, gelatin and various
Other substances may also be used, such as the animal sera of. Warm
Degree, protein selection and quench time depend on matrix material
It is a function of the type of probe used. Nonionic surfactant
The agent also quenches to reduce nonspecific binding.
Can be included in the wash buffer. After quenching the blot,
React with the probe. Generally, all reactions are
Performed in the presence of a hunting protein. Reacted blot
Then the buffer (in this case no protein
Thoroughly wash in). If the probe itself is radioactive
Yes, or is conjugated to an enzyme or fluorescent tag
Then this blot is immediately autoradiographed
Or react with a suitable substrate, or under UV light.
You can see it by yourself. Detect the presence of the probe-band complex
If a second or third reagent is needed for
Then incubate each reagent on the blot
After that, wash. The overlay technology is extremely sensitive
Very good, so very small amount of virus that exists in nature
Antigen can also be detected. Besides, these
The technique has also been used to analyze human sera from patients with various immune disorders.
Has been. One of the advantages of blots on gel is reuse
Or being able to repeat the reaction
is there. Once the signal is acquired and recorded, this blot
It can be deleted except for the probe, but the first protein pattern is
It can be left on the matrix. Erase
The filter further characterizes the components of the gel pattern
It can be reused for additional overlay analysis.
"Erase" lowers the pH to break down the antibody-antigen complex
Or blot the blot in urea or SDS.
By cubating and modifying the probe
Can be achieved. Probe van to find out detailed specifications
It is also possible to selectively decompose the complex. Calmodari
Calmodulin is Ca from the system⁺⁺By excluding Cal
It can be degraded from modalin binding proteins. this
The reaction is because the protein blot gives the first signal.
Can be done even after autoradiography
It The use of protein blots typically involves protein-protein
The purpose of investigating protein-ligand interactions
Or intermediate stage in immunological or biochemical analysis
To develop the production of immobilized polypeptide as a floor
Has a purpose. Both of these attempts
Has developed a lot of valuable uses. For example
is there.a : Analysis of protein-ligand conjugates DNA-protein and RNA-protein interactions are protein blots
Has been analyzed by. H₃ And H_Four Hist with
H₂ Conjugates are also illustrated. Epidermal growth factor acceptance
The body can hormone-overlay the mobile membrane pattern.
Confirmed by. Human epidermal cancer cell (A-431)
Cell membrane from SDS-polyacrylamide gel.
Run the gel on the DBM paper.
Lectroblot and epidermal growth factor followed by radiolabel
Antibody is used to quench the hormone and
Laid A very characteristic signal at 150 KD
was detected.b : Detection of enzyme subunits Detection of inactive enzyme on the protein blot was performed. example
For example, boil phosphodiesterase I in 2% SDS for 5 minutes.
Boiled and run on SDS-polyacrylamide gel.
The protein reacts with excess anti-phosphodiesterase I
Blotted into a nitrocellulose filter. Next
Incubate the matrix with a crude formulation containing active enzyme.
I had a masturbation. Inactive via vacant site of antibody
Activated and bound to divided subunits
The element was immobilized on this matrix. Then this
Trix reacts for enzymatic activity, causing immune complexes to
was detected. [0024]c: Affinity refinement of monospecific antibodies
Made Blots are monospecific antibodies
Is used for the purification of. The polypeptide is SDS-poly
Split on amide gel and on DBM or CNBr paper
Blotted to. This matrix should be
Overlaid with polyclonal antibody.
A single band containing the antigen-antibody complex was then applied to the mat.
It was cut from the lix, but the strip was
3) When incubated in buffer,
Pacific antibody was eluted. The elution probe is an immunological probe.
It could be used for cystic foci studies.d : Demonstration of cell-protein interactions To demonstrate specific whole cell-polypeptide interactions
A protein blot is used. Human plasma SDS
-Run on a polyacrylamide gel and apply nitrocellulose
Moved to Smatrix. Quenching the matrix
Normal rat kidney cells (NRK cells) after
I incubated it at
Immobilized polypeptide contained in a cell attachment
Was specifically bound to. Turn these cells into amino black
Therefore, when stained, two clearly distinguishable bands
It was found to be located in. These bands are five
Lonectin and newly discovered 70K Dentity
understood. [0025] The object of the present invention is to
Cationic charge modified Miku as a fixed matrix on the surface
From a Chromatographic Substrate with a Roporous Nylon Membrane
Solid phase for immobilizing macromolecules such as nucleic acids and proteins
The present invention relates to an assay system, wherein the solid phase is
By eliminating the above-mentioned various drawbacks of known materials,
Use a solid phase that immobilizes the polymer more efficiently.
It is to provide a solid phase assay system. Book again
Another object of the invention is to immobilize proteins from electrophoretic gels.
To provide a method of electrophoretically transferring to the lix,
This fixed matrix is better than the known nitrocellulose
It has good adsorption capacity and retention capacity,
It does not require high salinity. Even more
Another objective of Ming is that overlay operations can be performed efficiently.
Polymer blotting products and methods
To do. [0026] These various objects of the present invention
As a fixed matrix, essentially all of the wet surface of the membrane
Amount of charge-modifiable cationic charge repaired across
Charges composed of hydrophilic organic microporous membranes with decorating agents
Can be achieved by using a modified microporous membrane
It Preferably, the chaotic charge modifier has a molecular weight of 1000.
The above water-soluble organic polymer, each of which constitutes
At least one enzyme that allows the monomer to bind to the membrane surface.
A poxy group and at least one tertiary amine or
It has a quaternary ammonium group. Most preferred
Preferably, a part of the epoxy groups of the organic polymer is small.
At least one primary amino group or at least two diamines
Consisting of or containing few aliphatic amines with primary amino groups
At least one secondary amino group and carboxyl or hydroxy
Secondary charge composed of aliphatic amine having droxyl substituent
It is bound to the load modifier. The charge modifier is also a resin having a molecular weight of 500 or less.
Bound to the membrane via a cross-linking agent consisting of an aliphatic polyepoxide
Can be aliphatic amines or polyamines
Yes. The preferred microporous membrane is nylon. The phone
The load-modifying microporous membrane is used in the form of a reinforced laminate membrane.
May be used, and preferably a polymer microporous membrane is used.
Use in the form of a porous reinforced web impregnated with
It The immobilization matrix of the present invention is essentially a wet surface of the membrane.
Charge modification of cationic charge modifiers that are bound throughout
Organic microporous possessed in sufficient quantity for decoration
Consisting of hydrophilic charge-modified microporous membrane
It Microporous membranes and cationic charge modified membranes
It is known in the field of filtration of bodies, such as liquids. Fluid filtration
Cationic charge modified microporous membrane in the field
Please refer to EPC publication No. 00668
14 and EPC Publication No. 0050864.
It Here, high-purity water (18 m
A description of using a charge-modified membrane for filtration of eg ohm-cm resistivity.
And; charge-modified membrane for filtration of parenteral or body fluids
There is a description using. Furthermore, these filtration membranes
It is also known that can be enhanced by various means. Further, commercially available nylon microporous
Filter membranes are available from Pall Corp., GlenCove, NY as “ULT
IPORN₆₆And N₆₆POSIDYNE "provided under the trade name
And the latter is a filter membrane with cationic charge modification.
is there. Furthermore, at AMF Inc., Cuno Div.,
Charge modified nylon microporous filter
The membrane is sold as "ZETAPOR" (trade name). here
The term "microporous membrane" used in
A substantially symmetrical and isotropic porous film, which has a pore size
Is at least 0.05 micron or more or the initial
The bubble point (IBP) is 120 psi (8.37 Kg / cm) in water.² )
It means a porous film having the following pore size. this
Pore size is about 1.2 microns or less, about 10 ps as IBP
i (0.69Kg / cm² ) May be larger. For "target"
The term says that the pore structure is substantially the same on both sides of the membrane.
means. The term “isotropic” means that the membrane is
It is meant to have a uniform pore structure. The membrane is preferably hydrophilic. micro
The term "hydrophilic" to describe a porous membrane is a membrane
Means to absorb or adsorb water. In general,
Such hydrophilicity has a sufficient amount of -OH groups, -COOH groups,-
NH₂ Groups and / or similar functional groups are present on the surface of the membrane
Occurs when Such functional groups absorb water on the membrane and
/ Or promote adsorption. Hydrophilicity of fixed matrix
Gender is a necessary element of the present invention. The hydrophilicity of the membrane itself
Providing a suitable binding force of the charge modifier to the microporous membrane of
To serve. A preferred microporous membrane is "nylon" (commercial
It was created from the (name). The term "nylon"
Copolymers and terpolymers containing repeating amide groups
Means a film-forming polyamide resin that includes the body
It Generally, various nylon or polyamide resins
All are copolymers of diamine and dicarboxylic acid
Or a homopolymer of an amino acid lactam, wherein
Wide range of crystalline or solid structures, melting points and other physical properties
Has changed to. Suitable for use in the present invention
Nylons include hexamethylenediamine and adipic acid
Copolymer (nylon 66), hexamethylenediamine and ce
Copolymer with Basic Acid (Nylon 610) and Poly-O-Ca
It is a homopolymer of prolactam (nylon 6). On the other hand, these suitable polyamide resins are
Chilen (CH₂ ) To amide (NHCO) group ratio is about 5:
1 to within about 8: 1, more preferably about 5: 1
The range is about 7: 1. Nylon 6 and Nairo
66 to 6: 1 respectively, nylon 610 to 8: 1
It is a ratio. Many brands of nylon polymers are available
Therefore, the molecular weight ranges from about 15,000 to about 42000.
In addition, various other characteristics are commercially available. Polymer
The most preferred type of chain-constituting unit is poly
Hexamethylene adipamide, or nylon 66
Therefore, the molecular weight is preferably about 30,000 or more. Contains no additives
Polymers are generally preferred but antioxidants or similar
The addition of the additive is preferred under certain circumstances. Suitable membrane
Substrate prepared by the method described in US Pat. No. 3,876,738
To be done. Another method is European Patent Application No. 0005536.
No. Furthermore, for example from Pall Corp.
Provided "ULTIPORN₆₆(Nylon, trade name), Millipor
"Durapore" (polyvinylidene fluoride
Id, brand name), cellulose acetate commercially available from other companies
/ Any of the nitrate films used in the present invention
Suitable for cationic charge modification for The above-mentioned US and European patent applications
The feature of the nylon membrane is that it has an isotropic structure.
Highly effective surface area, narrow pore size distribution and
Consists of a controlled size pore with a cut pore volume
It has a fine internal microstructure. For example, typical
0.22 micrometer nylon 66 membrane (polyhexamethylene
Adipamide) is approximately 45-50 psi (3-3.5 Kg / cm)² ) IB
P, 5psi (0.35Kg / cm² ) (47mm diameter disc)
Provides a water flow rate of 70-80 ml / min at
T, nitrogen adsorption) is about 13m²/ G, the film thickness is about 4.5 ~
4.75 mils (114-120μ). Charge modifier is micro
Bonds across virtually all wet surfaces of the porous membrane
There is. The term "bond" refers to the charge modifying agent (s)
/ Or they are well connected to each other and a large amount under the conditions of use.
Means that it is not extracted. "Wet
The term "substantially all of the surface" refers to outer and inner pores.
All of the surface is due to fluid flowing through the membrane or
Means that the membrane becomes wet when immersed in a fluid
It One suitable charge modification that can be used in the present invention
The agent is described in Ostericher et al., And their molecular weight is about 1000.
The above water-soluble organic polymer in the monomer structure
Contains at least one epoxide capable of binding to the membrane surface.
Such as capable of providing side substituents and cationic charge sites
At least one tertiary or quaternary ammonium group
Existing. This charge modifier is a polyamide-polyamid
Nepchlorohydrin type cationic resin, especially
Preferred are the resins described in U.S. Pat.
  U.S. Pat.No. 2,926,116 U.S. Pat.No. 2,926,154 U.S. Pat.No. 3,224,986 U.S. Pat.No. 3,311,594 U.S. Pat.No. 3,332,901 U.S. Pat.No. 3,382,096 U.S. Pat.No. 3,761,350 This polyamide-polyamine epichlorohy
The drin type cation resin is "Polycup 172, 1884, 2002
Is S2064 "(Hercules trade name); Cascamide Resin p
R-420 "(Borden company name); or" Nopcobond 35 "
(Nopco's trademark name). The best one
"Hercules R4308" (trade name)
The nitrogen atom forms part of the heterocyclic group and the methylene part
It is bonded to the reactive epoxy group through the position. The monomers that make up the "R4308" resin are as follows:
General formula 4 [Chemical 4] have. On the other hand, "Polycup 172, 2002" and "P
olycup1884 "resin is composed of the following general formula
5 [Chemical 5][Wherein R is methyl or hydrogen (Polycup 172 and
R = H in 2002; R = CH in Polycup 1884₃ ]
ing. Most preferably, it is water-soluble with a molecular weight of 1000 or more.
When using an organic polymer as a charge modifier, this
Enhance the cationic charge of the secondary charge modifier and / or
The microporous surface of the primary charge modifier and / or its
Secondary charge modifier for enhancing the binding force to itself
Is to use. This secondary battery used in the present invention
The load modifier is selected from the group consisting of: (1) At least one primary amine or at least two
Aliphatic amines having a secondary amine moiety of: and (2)
At least one secondary amine and carboxyl or
Aliphatic amines having a hydroxyl substituent. Preferably
This secondary charge modifier has the general formula 6 [Chemical 6] [In the formula, R₁ And R₂ Is an alkyl group having 1 to 4 carbon atoms,
x is an integer of 0-4. R₁ And R₂ Both are d
It is preferably chill. ] With a polyamine
is there. Suitable polyamines are: Ethylenediamine H₂ N- (CH₂ )₂ -NH₂ Diethylenetriamine H₂ N- (CH₂ )₂ -NH- (CH₂ )₂ -NH₂ Triethylenetetramine H₂ N- (CH₂ -CH₂ -NH)₂ -CH₂ -CH₂
-NH₂ Tetraethylene pentamine H₂ N- (CH₂ -CH₂ -NH)₃ -CH₂ -CH₂
-NH₂ The most preferred polyamine is tetraethylene pentamine
is there. Alternatively, the aliphatic amines used in the present invention
Is at least one secondary amine moiety and one carboxylate
It may have sil or hydroxyl substituents.
Examples of such aliphatic amines include γ-aminobutyric acid (H₂
NCH₂ CH₂ CH₂ COOH) and 2-aminoethano
(H₂ NCH₂ CH₂ OH). This 2
The secondary charge modifier is an epoxy substituent of the polymer primary charge modifier.
By binding to a portion of the microporous membrane
Are combined. Of the primary and secondary cationic charge modifiers used
Amount enhances the positive capture potential of microporous membranes
Enough to handle Such amount was used at that time
There is a great dependence on the types of charge modifiers. In a broad sense, the above-mentioned primary and secondary cation electrodes are
The load modifier is a hydrophilic organic polymer microporous membrane, such as
To be bonded to nylon, with epoxy substituents
Of primary cation charge modifier that binds to membrane structure via
It is combined by applying an amount to the membrane. Preferably
The method is as follows: (a) an aqueous solution of a primary cationic charge modifier for the membrane.
And (b) contact the membrane with an aqueous solution of the secondary charge modifier.
Touch it. The contacting process is followed by the following sequence: process (a).
Step (b) or vice versa. But first the membrane 1
Contact with a secondary cationic charge modifier, followed by secondary charge modification
Contact with the agent produces the smallest extractables.
The best results. In another embodiment of the present invention, the secondary charge is
Fats with a molecular weight of 500 or less using a modifier as a charge modifier
This microporous via a group polyepoxide crosslinker
It may be adapted to bind to the membrane. Preferably
This polyepoxide has a molecular weight of about 146 to about 300.
Alternatively, it is a tri-epoxide. Such polyepoxy
Viscosity at 25 ℃ is less than about 200 centipoise.
(At the time of release). In view of the purpose of acting as a crosslinking agent,
This epoxide is a monoepoxide such as glycidyl.
Things like ruthels are unsuitable. Similarly, three
Polyepoxides that provide more than one epoxy group are
There was no benefit, in fact, due to steric hindrance
Coupling reaction of lever polyepoxide is restricted
This is theoretically clear. In addition, it is cationically charged.
The unreacted epoxide groups are
Its presence may adversely affect the final product. The most preferred polyepoxides are of the formula [Chemical 7][In the formula, R is alkyl having 1 to 6 carbon atoms, and n is 2
Is ~ 3]. Non-epoxy part
The number of carbon atoms in-(R)-is 6 or less, therefore
The polyepoxide is water or a mixture of ethanol and water,
For example, it is soluble in a mixed solution of 20% ethanol or less. this
Although those with more carbons are functionally useful,
It is necessary to use a polar solvent when using it. Toxicity and ignition
It is necessary to pay attention to the characteristics and environmental pollution. The aliphatic aminopolyamine charge modifier is
 (a) contacting an aqueous solution of this cationic charge modifier with a membrane,
And (b) bringing an aqueous solution of the polyepoxide crosslinking agent into contact with the membrane.
To bind to the microporous membrane. Contact
May be step (a) followed by step (b) or vice versa
It Such a contacting step also causes the membrane to be charged with a charge modifier and a polyepoxide.
Includes a method of contacting an aqueous solution of a mixture with a xide crosslinker.
Including. However, contacting the membrane with a cationic charge modifier
Then, the treated film was contacted with the polyepoxide crosslinking agent aqueous solution.
Minimize flashout time when touched
It is most preferable from the viewpoint. But maximize charge modification
To mix the membrane with a charge modifier and a polyepoxide crosslinker
It is better to contact them with an aqueous solution of the substance. Contacting this microporous membrane with this aqueous solution
Then, dry it in a tensioned state to prevent shrinkage.
Good to crosslink. Drying the membrane under tension
It is described in US Patent Application No. 201,086. general
Any tensioning means may be used, for example
It may be wound along a dry surface, like a flat drum.
Biaxial control is preferred, pulling the membrane into the expanded frame
Is best. Tension applied does not reduce size
It is preferable. Final drying and crosslinking temperature is about 120 ℃
It is often done at 140 ° C, at which temperature the film deteriorates
Drying time can be minimized without becoming brittle. Finish
The sharp film is rolled and stored at room temperature. The charge-modified microporous film also has a strong
Can be used as a chemical and / or laminated membrane
And is preferably substantially cationically charged
Molecular microporous membrane creates one porous reinforced web
It can be used in impregnated form. Reinforced laminate film and por
Russ reinforced webs are described in US Pat.
There is a listing. Such impregnated webs should have a sufficient amount of casting liquid.
Is cast on a porous reinforced web and the casting liquid is placed on top of it.
After making a web that holds the
Good to make. This reinforced web is filled with casting liquid during casting.
Wet with the casting solution to maximize the impregnation of the
And precipitation of polymer membranes such as "nylon"
Is it a porous material that will adhere strongly to the web?
Consists of However, this web can be wetted with casting liquid
That is not a requirement. Non-wettable web
In the case of
Limited to face only, but nevertheless
Impregnation of solution into web and adhesion of film to web
Attached to the membrane by. For example, such wettable and non-wettable reinforcement
The web is made of extruded plastic filament net, paper.
Various types of non-woven fabrics including par and similar materials
It can be made from bituminous nets. Strong non-wetting property for casting liquid
The synthetic web is made of polypropylene or polyethylene.
Comprising a microporous nonwoven web made from eel fibers
You can Some suitable wetness-enhancing webs are monofilaments.
Filament or multifilament yarn
Polyester consisting of woven or non-woven web
Some of them are made of monofilament and the monofilament at this time is open
Structures with low pressure loss are preferred
Woven polyamide fiber web, aromatic polyamide and
Lulose, regenerated cellulose, cellulose ester, cell
Other relatively like loose ether, glass fiber etc.
Woven and non-woven webs made of polar fibrous products
Which is included. Cellulose and synthetic fiber filter paper
Par is also a reinforced web and perforated plastic sheet.
And open mesh expanded plastic
Can be used. Substrate is relatively open and remarkably open
In the case of a single structure, even if the fibers are substantially
The substrate is impregnated with the membrane material, even if
It can be done. Like polypropylene or polyethylene
Non-wettable materials ensure that they have a sufficiently open structure.
If so, it can be impregnated by the membrane. Preferred embodiments for making impregnated reinforced webs
Casts a sufficient amount of casting liquid onto the porous reinforced web
Then, create a web that holds the casting liquid on it.
It Helps this coating penetrate into the web
In order to reduce the viscosity of the liquid sufficiently, and to form a coating
Effective temperature to eliminate all entrained air in the web
When the coating liquid is removed from the web under the conditions of temperature, pressure and time.
It is made to permeate with under roll. Such temperature, pressure and
The time and time conditions are the reinforced web used, casting liquid roller, etc.
Depends heavily on the type of. These terms are posted on the website
Observe the liquid seepage condition, pinholes and bubbles in the final product.
The person skilled in the art can easily determine the
You can The coated web is then placed on top of
Applying the casting liquid to the casting process to retain additional coating liquid
Form a finished web. Then quench this web
Quench in the bath to form an impregnated web, then
The outer membrane is laminated to this web. After forming this reinforced film, it is used in the present invention.
To form a cation-charge modified microporous film
It can be processed according to the method described above for
It This cationically charge-modified microporous membrane is
Conventionally, nitrocellulose and DBM paper have been used
In the same manner as described above, immobilization markers in polymer blotting
Used as a trick. However, the charge repair of the present invention
Decorative microporous membranes are better than traditional fixed matrices
Has much greater capacity. For example, the charge repair of the present invention
Decorative microporous membrane is at least 480 μg / cm² Protein
It has binding capacity, but that of nitrocellulose is about
80 μg / cm² Nothing more. The fixed mat in the present invention
It is common for lix when using nitrocellulose.
The use of methanol in transfer buffer is a prerequisite
is not. Moreover, nits used in connection with DNA
Rocellulose immobilization matrix is a high concentration of salt in the buffer.
As a result of the required minutes, high currents of up to 5 amps are required
However, the heat generated at this time adversely affects the DNA. Book
High salt when using the charge-modified microporous membrane of the invention
Sub-concentrations are not required and therefore in the order of milliamps
Electrophoretic analysis is possible. The transferred electropherogram overlay is
Same as done in other traditional fixed matrices
The same can be done with the fixed matrix of the present invention as well.
It is possible. This matrix, like any other matrix
Quench the latent binding sites remaining on the substrate
To minimize non-specific background by
Must. Charge modified micros used in the present invention
Porous membranes have a high affinity for proteins, which
The usual quenching methods are not enough. Of the present invention
The charge-modified microporous membrane is phosphate buffered saline (PB
45% to 50% overnight in 10% bovine brain serum (BSA) in S)
Incubation at ℃ effectively quenches
It turned out that it is possible to hunt. Hemoglobin
(45-50 ° C, 1% in PBS) is also available for quenching
It turned out to be effective. This invention uses a chromatographic substrate to
Especially useful in connection with electrophoretic transfer to
Fruity, but also matrix from chromatographic substrate
Mass transfer of liquid as an initiating force for the elution of macromolecules into
Like movement by transportation and movement by diffusion
It also applies to various blotting techniques. To this invention
Thus, the cationically charge-modified membrane is simply polyacylated.
Derived from lylamide or agarose gel
Can be used as a fixed matrix for macromolecules
As well as thin layer chromatography or high voltage
Other chromatographs such as the products of pressure paper electrophoresis.
Or used directly for spotting or solid-phase analysis.
Polymers derived from solutions containing macromolecules
Can also be used as a fixed matrix for
It [0050] The present invention will be described in detail below with reference to examples.
The invention is not limited to only these examples. [Example 1] A.Creation of microporous membrane Nominal surface area about 13m²/ G, initial bubble point approx. 47 ps
i (3.3 Kg / cm² ), And Foam-All-Over-Pint
Is about 52 psi (3.6 Kg / cm² ) Indicating a nominal rate of 0.22
US Patent for Chromometer's typical nylon 66 (trade name) membrane
Nylon 66 according to the method described in Japanese Patent No. 3,876,738.
(Trade name, Monsanto Vydyne 66B) 16% by weight, methano
Composition of 7.1% by weight and 76.9% by weight of formic acid,
Quench bath composition of 25% by volume of methanol and 75% by volume of water (required
If necessary, use the method described in U.S. Patent No. 3,928,517.
Regeneration), casting speed 61 cm / min, quench bath temperature 20
Created at ° C. Apply to casting drum rotating in bath
Cast the film directly below the quench bath surface by applying a cloth
From the drum to draw an arc of about 90 ° from the application point
Peeled off. This film forms a shallow catenary when picked up
It was a self-supporting film. Part of this uniform opaque film
Minutes for 30 minutes in forced convection in an air oven at 80-90 ° C,
It was dried in a pressed state to resist shrinkage. B.Fabrication of charge-modified microporous membrane 1. Polyamide-based membrane samples (dry sample and undried sample)
Polyamine epichlorohydrin resin (trade name, Herules
 1884) in a 4 wt.
I reached the equilibrium. Wash the treated membrane sample to remove excess resin
Clean and then hold it on the drum at 110 ℃, about 3
Dry for minutes. Processed film sample flow as follows
And bubble point characteristics were compared, but for treated and untreated samples
It turned out that the pores and surface
I found the line maintained. The results are shown in Table 1.
It was [0053] FAOP (Foam -All-Over -Poin
t) is in accordance with ASTM D-2499-66T, IBP (InitialBubb
le Point) and then air is passed through the wet film sample
Increased air pressure until flowing and placed between regulator and sample holder
It will be 100cc / min when read by the flow meter
I'm sorry. FAOP is directly proportional to the average pore diameter of the sample film
To do. Morphological and liquid mechanics controlling mechanical sheaves.
The characteristic of the treated film is
It can be seen from Table 1 that the characteristics match those of the nylon membrane.
Light 2. Same as for other film samples prepared in the same manner.
The characteristic values were compared, but here, "Hercules R430
8 "resin (brand name, diallyl, nitrogen-containing compounds
Free radical polymerization resin,
2% by weight as solids)
Treated in a bath of 0.5 and then 0.1% by weight of tetraethyl
Topcoat with pentapentamine, drying, crosslinking, washing with water and re-drying
Dried The results are shown in Table 2. [0056] The surface area of the treated and untreated sample membranes is essentially
Unchanged; tensile strength increased with treated
However, the growth was slightly decreasing. The processed sheet is flexible
Improved; cracks when folding untreated sheet
Raised and torn. [Example 2] Wet Mi prepared in Example 1A
Laminate two layers of chloroporous film together, moisture 20 to 25
% To dry. So moist and swelling conditions
The underlying membrane is more effective than the fully dried membrane.
It was found to absorb load modifiers. Then this two-layer film
Was dissolved in 1.25% by weight of "Hercules R4308" (trade name) resin.
It was placed in the liquid. The pH of the bath was 10.5. The bath is "Her
cules R4308 ”resin 17.17Kg from the initial 20% by weight to 5 times
Prepared by diluting to volume%. Then 5N-N
The pH was raised to 10.5 by adding aOH. Then add this solution
D. having a resistance of 150000 ohm-cm or more I. Volume ratio with water 2.
Diluted 5: 1. The total volume of bath solution is 60 gallons (227 L)
Met. The membrane is "Hercules R" at an angle of 30 ° from horizontal.
4308 "bath, charge modifier penetrates into the membrane
Prevents inclusion of bubbles in the membrane
It is. The membrane is 121.9 cm long in this bath at a speed of 76.2 cm / min.
It was processed over a period of time. Remove from bath and remove excess water
I wiped off the bottom surface. The membrane in a secondary charge modifier bath
Soaking for 3 minutes in a stream of cold air before introduction. The
The bath is D.I. I. Water (at least 150000ohm-cm resistance) 227L
Adding 0.0513 Kg of tetraethylenepentamine
Prepared by The pH was about 9. Immersion condition is primary
It was identical to the charge modifier bath. Then take up the membrane
Wrapped around the reel. Picking row with wet film
The le was stored for at least 3 hours. Then roll 121
The reaction with the charge modifier was completed by drying at 3 ° C. for 3 minutes.
The membrane was then washed and the extraction level checked. [Example 3] Made according to Example 1A
Microporous “nylon” (trade name) membranes with “Hercu
les R4308 ”primary charge modifier (adjust pH to 10 with NaOH)
Preparation) and then with a polyamine secondary charge modifier
Processed. As shown in Table 3 (Table 1), each film
There is little or no difference between the flow characteristics of the
It was. [Table 1][Example 4] Different charge primary modifiers,
Especially polyamide / polyamine epichlorohydrin resin
Complementary performances are compared and the application level and pH are optimized.
"Hercules resins R4308" "Poly cup 17"
2 "(commercially available at pH 4.7) and" Polycup 2002 "(solid content 2
7% by weight, marketed at pH 3.0) (all above are trade names)
The following tests were carried out using The results are shown in Table 4. [0062] [Example 5] Made according to Example 1A
"Nylon" (trademark) microporous membrane
The washed, pulled, and dried membranes were treated with 18 megohm-cmD. I.
80:20 mixture of water and high-purity ethanol (pH is 6.1 to 6.4)
1,4-butanediol diglycidal prepared using
It was immersed in an ether solution (solid content 2% by weight). A solution?
The film was pulled up and drained for about 1 minute. Then 18 megoh
m-cmD. I. Prepared with water, tetra-hydrate showing pH 11.2-11.4
Immersion in ethylene pentamine solution (solid content 0.5% by weight)
did. Pull up the film, drain it for about 1 minute, and then spread it.
Dried at 130 ° C. for 5 minutes. [Example 6] "Nylon 66" (trade name)
16% by weight, formic acid 78.04% by weight and methanol 5.96% by weight
Was spunbonded using a casting solution consisting of
"Ree may 2550 Polyester" (from duPont Corporation
Apparent pore size with a reinforced web consisting of
Nylon containing with a microporous membrane of about 0.65 microns
A soaked web was created. First side of this microporous membrane
Is contacted with the impregnated web to soak the two-layer joint.
Forming wet contact lines and similarly impregnating webs
Place the second side of the microporous membrane on the opposite side of the
It was This three-layer laminated film is applied to both sides of the laminated film.
"Teflon" (commercial
(Title) Dried on coated steel drum. Surrounding the drum
Infrared heaters were arranged at intervals across the.
After that, this reinforcing laminate is treated with a cationic charge modifier.
Treated according to Example 1B. [Example 7] A. Materials and methods Protein Sample Preparation: Red Blood Cell Ghosts at Fairbanks, G., Ste
ck, T. L. And Wallach, D. F. H. [(1971) Bioche
mistry 10: 2606 to 2617]₁ Mace
In the pleural cavity above the heart puncture
It was prepared from blood accumulated in. DeCamilli, P., Ueda,
T., Bloom, F. E., Battenerg, E., and Greengard, P.
 (1979)Proc.Natl.Acad.Sci.USA.:76, 5977
To bovine brain skin homogenate according to the method described in
Was prepared. Protein Standard, Protein A and concanaval
Radioiodination of in A is described by Fraker, P. J., and Speck, J.
C. (1978) 〔Biochem.Biophys,Res.Commun.:80, 84
9-857, 1,3,4,6-tetrachloro
Prepared from -3a, 6a-diphenylglycoluril
However, the protein was dissolved in PBS (1 mg / ml). 5
-Ml Biogel P-2 (Bio-Gel, trade name) or
 Either on a 0.4-ml AG-1-X8 column¹²⁵I-
Free from labeled protein¹²⁵I was isolated. Special activity
Sex 10⁶ ~Ten⁷ cpm ug^-1Always obtained from protein. Polyacrylamide gel electrophoresis analysis:
2% (w / v) SDS (as final concentration), 2% (v / v) β-
Mercaptoethanol, 10% (v / v) glycerol, 0.1
(w / v) Bromphenol blue, and 100 mM Tris-H
Stabilized with Cl containing pH 6.8 buffer. The sample
Boil in mixture for 3 minutes, then Laemmli's system
(Laemmli, U. K. (1970)Nature (London):227, 680
~ 685] on 10% polyacrylamide slab gel
Was resolved. Electrophoretic migration: After completing electrophoresis,
Gel (or part of it) from Scotch-Brite
Place it on a table and move its surface cold (8-10 ° C).
Buffer) buffer (15.6 mM Tris-120 mM glycine,
pH 8.3, with or without 20% (v / v) methanol
Rinse. Tris (41 mM) -boric acid at pH 8.3
A (40 mM) buffer was also tested. These two types of buffer cis
No difference was observed between the Thames. Then real
A cationic charge-modified microporous membrane of Example 2 (hereinafter CM
MM) or nitrocellulose (both are general
Fixed matrix "IM") on transfer buffer
Float and moisten and place on gel, inside or after IM
That no air bubbles have been carried between the person and the gel
confirmed. This IM is then passed to the second Scotch-Brite package.
I covered it with Do. Minutes by Scotch-Brite pads
A group of these gel-IM assemblies that have been eclipsed are for simultaneous migration
Can be arranged continuously. This assembly is placed between the plastic grids.
Place, then place the grid in 4 liters of cold transfer buffer
In a tank made of "Plexiglas" (trade name) ... towards the anode
I just inserted it with IM.Anal.Biochem. (19
80): 102, forms a sequence similar to that described in 459-471.
The platinum electrode was fixed to the wide side wall of the tank. Anode and power
The sword interval is 8-cm, and the voltage generated during electrophoretic migration is
The field was observed to be averaging. During the move operation,
Elution from the buffer composition gel converted as salts,
The current increased when the voltage was kept constant. Standard input
Since it was used (Buchler Model 3-1500), a constant current (2
It is possible to move it at 00mA) and gradually decrease the voltage.
It was sometimes observed that it was practical
It was In a typical 2 hour trip, the voltage is, for example, 42 to 30
I dropped to the bolt. This voltage drop is due to migration buffer and gel
That can be avoided by preferential balancing between
It This method also avoids gel swelling during migration,
This swelling is always present when the acrylamide concentration is ≧ 10%
It exists. IMs can be used immediately after moving
Allow or dry Whatman 3MM chromatography pellets
It can also be stored and used between the sheets of the supermarket.¹²⁵
In experiments where migration of I-labeled protein was quantified, gels were used.
And IMs for both Kodak XAR-5 and DuPont Cronex
Using Lightning Plus Strengthening Screen (at -70 ° C)
Auto-radiograph of post-transfer
It was measured by This emission band is the gel and IMs
Triggered by Beckman Biogmma 2 counter
Was done. Transfer treatment with antibody and lectin
Rationale: To eliminate unspecified background binding, I
Ms must be quenched. Nitrocellulo
2% (w / v) BSA or 1% (w / v)
In 10 mM PBS (pH 7.4) containing either hemoglobin
It is sufficient to incubate this filter for 1 hour at room temperature
Is. 10% BSA for CMMM incubation
Or phosphate buffer containing either 1% hemoglobin
CMMM, IMs for 12 hours at 45 to 50 ℃ in buffer solution (PBS)
It was found that culturing was sufficient for quenching.
It was. IMs that have been quenched are 2% BSA or 1%
Suitable Riga in PBS containing any of the moglobin
At room temperature (eg antibody, protein A, lectin) at room temperature
For at least 5 times, then at least 5 with 50-100 ml PBS.
Washed twice. All solutions used in the above operation were
It contained muazide (0.05% w / v). Washed IMs
Were autoradiographed at -70 ° C as described above.
It was B.Result 1. Binding of macromolecules to IMs (a) to IMS¹²⁵Comparison of adsorption of I-labeled BSA
In order to do so, a joining test was conducted. Square IM material at 1:00
For various concentrations¹²⁵I-labeled BSA (0.01-5%
w / v in PBS) solution. Cultivation
An equal amount of tracer (177000
± 6100 cpm ・ ml^-1; Estimated amount 180ng ・ ml^-1)of ¹²⁵I-la
Belled BSA was included. After incubation
Then, the IMs were washed 5 times with 5 ml PBS and counted.
It was The count is the combined BSA (ug ・ cm^-2).
The ability of CMMM to bind BSA is that of nitrocellulose
I was always over capable. This is due to the high concentration of BSA
It is especially noticeable when used, for example with 5% BSA
IMs is 480ug ・ cm^-2In contrast, Cellulose is 80ug ・ cm
^-2Met. This is shown in Fig. 1, where the CMM is
M is ● (black circle) and Nitrocellulose is ○ (white circle)
Displayed in. (B) 1 cm² CMMM, Nitro Cellulo
Of unmodified nylon and the membrane of Example 5 with hemoglobin
(0-5% in PBS) or BSA (0-10% in PBS)
Were incubated at various concentrations. Each fill
1 ml of culture solution was cultivated and rotated (2 h, RT ° c). Next
so ¹²⁵50 μl of the I-labeled protein mixture was added to each well.
In a well [50 l = 5000 cpm IgA; 5
000cpm Calmodulin; 5000cpm BSA]. Like this
Incubate the filter at 25 ℃ for 1 hour
Was washed 5 times with 1 ml PBS and counted. back
Ground (85cpm) is subtracted from the result, and Fig. 1a (BS
A) and FIG. 1b (hemoglobin). For example, 1%
In BSA, the signal of the radioactive binding protein is nitro
Cellulose CMMM, unmodified nylon and membrane of Example 5
Only 98%, 48%, 94% and 77% if used
It decreased respectively. Next, IMs that are not quenched
Between 1% "Trition X-100" (trade name, nonionic
Wash in detergent) to determine how much radioactive protein
It was measured whether it was removed. As a result, 40 from CMMM
% From the IM of Example 5, about 80% from the unmodified membrane
92% and 92% removed from nitrocellulose
It turned out that These results show that protein and IMs
It clearly shows that charge modification contributes to the interaction.
It (C) 2 cm of IMs² 5 using 4 squares
0000 cpm of³²P] and label (in vitro)
Human β-globin mRNA [Nucleic Acid Resea
rch,Ten: 5429 (1982)]
It was Then the count during washing reaches the background
Washed with PBS until Then attach it to the filter
The amount of labeled mRNA present was measured. Commercial nitro
Cellulose and cellulose acetate squares are 100 cpm
Although the binding amount was as follows, two CMMMs (porosity
In 0.45 and 0.2) respectively, the binding amount was about 30,000 cpm. 2. Electrophoresis of proteins from gel to filter
Dynamic movement Buffer conditions similar to those described by Towbin et al. (Ie 15.6 mM
Tris, 120 mM glycine, 20% (v / v) methanol, pH 8.
Using 3),¹²⁵Electricity of I-labeled BSA (~ 200ng)
Migration (30V, 1 hour) by electrophoresis method was tested for IMs.
I tried When methanol is omitted from the buffer, CM
In each case of MM and nitrocellulose,
The separation increased to 30% ~> 60% and ~ 50% of the load respectively
It was However, in the absence of methanol, C
Corresponding to the use of MMM and nitrocellulose
Elution increased by 30-> 60%. However, in the absence of methanol
In case¹²⁵I-labeled BSA has at least 5 layers of nit
Rocellulose (on this surface there is a 15-8% reduction in load)
Detected) passed; however most of¹²⁵I-la
Belled BSA (> 60% of load) is the first CMMM file
On each successive filter remaining on the router
<1% was detected. Other protein standards
(Phosphorylase b, fertin, ovalbumin, carbonic an
hydrase and soybean trypsin inhibitor)
Similar results were obtained. Performed in the absence of methanol¹²⁵
Representative results for the transfer of I-labeled BSA are shown in FIG.
~ Shown in FIG. FIG. 2 shows various variables.¹²⁵I-labeled BS
Quantify the transfer of A to CMMM or nitrocellulose
The results are shown below.¹²⁵Increase the concentration of I-labeled BSA by 4 types
10% when added (estimated at 150, 300, 514 and 1400ng)
Run on SDS-polyacrylamide gel. Electric swimming
After the movement, eight CMMM layers that continued one lane series
Move to (●, ■, ×), and nitro cellulos for others
It was moved to 10 consecutive layers (○, □, △) of the fabric. Move
After completion (constant 32V, no methanol, pH 8.3, Tris
-2 hours with boric acid), count the gels and IMs
did. The amount of BSA eluted from each gel lane (○, ●) is
Corresponding gel post-transfer residual release
Calculated as the difference between radioactivity and loaded radioactivity. Not yet
Count BSA (△, ×) is the radioactivity eluted from each gel
CMMM or nitrocellulose filter corresponding to the property
Calculated as the difference between the radioactivity recovered on (□, ■)
It was FIG. 3 shows the highest load BSA used (1400
ng) to eight CMMM filters (●),
On 10 nitrocellulose filters (○)¹²⁵
I-labeled BSA is shown. Arrow 1 is eight CMs
The total amount recovered on the MM layer is shown. Arrow 2 is a nitro cell
The total amount recovered on 10 layers of sucrose is shown. FIG. 4 shows the first CMMM filter, the first
Nitrocellulose filter and 10 continuous nitrocells
It is a comparison of the BSA recovered on the loin layer. Shown in Figure 2
The results obtained from the experiments were
The amount of BSA recovered on all 8 layers of CMMM.
Married The value was 100% recovery. Three types of BSA
For lower concentrations of, the first CMMM layer (stripe
Is a total of 10 layers of nitrocellulose (hatch bar)
Absorbed about the same amount or more. In any case
The amount of BSA recovered on the first CMMM layer is the first
Collected on a rocellulose filter (strip bar)
Significantly larger (4x) than that of 2, FIG. 3 and FIG. 4 show (1) CMMM 1
If two or two layers were used, the proteins loaded on the gel
Show that you can count over 80% of white
(Figs. 2 and 3); (2) 80% of IM-bound BSA (low low
Or 50% (high load) on the first layer of CMMM
It was recovered (Fig. 4); and (3) CMMM
No improvement in recovery was observed even if 3 or more layers were used
(Fig. 3). Using 10 layers of nitrocellulose
Similar results were not obtained (Figs. 2 to 4). Nitro
Not counted when using cellulose¹²⁵I-label
BSA (probably lost in buffer)
> 25% of the standard, but in the case of CMMM it is about ≦ 15%
It was degree. 3. Retention of transfer proteins on the filter Once adsorbed to CMMM, the protein becomes very good
The retention was proved by the following experiment. SDS
-Run on polyacrylamide¹²⁵I-labelling
BSA charges either CMMM or nitrocellulose.
It is moved electrophoretically. After moving this IMs to the following conditions
Incubated with one of: (1) PBS
 15 minutes in 0.5% glutaraldehyde, (2) 25% isop
Lopanol in 10% acetic acid for 1 hour and (3) PBS alone
1 hour in the taste. Then rinse the IMs several times in PBS.
Then 0.1% "Triton X-100" in PBS (quote
It was left to stand in the (brand name) for one day and washed. Label on each IM
The amount was measured both after transfer and after washing. Nitrocellulo
In case of¹²⁵80% of I-labeled BSA is unspecified
Washed out from incoming IMs; glutaraldehyde or acid-
At least 1.5 ~ on IMs after alcohol treatment
More than double counts were retained. When using CMMM
> 65% of the original count is retained without the fixative,
With increasing fixer, this value reached> 90%. That
The retention of protein in CMMM depends on the pH of the washing solution (2.0 to 8.3).
It was found that even if it fluctuates, it is not actually affected. 4. Transfer overlay What Overlay Technology Uses Moving Patterns
Even before that, the residual potential on the filter
Binding sites minimize unspecified background
Must be quenched for. CMMM is 45
At ~ 50 ° C, in 10% BSA in PBS overnight.
If you incubate, it will be effectively quenched. Than
At low temperatures (eg 37 ° C), lower concentrations of BSA, or at 50 ° C
Incubate the filter with the above solution for a shorter time.
In basation, when these probes are used
This is good because it creates a high background during the overlay.
Not good. Hemoglobin (45-50 ° C, 1% in PBS)
Also for quenching CMMM transfers
It turned out to be effective. The protein pattern overlay is either an antibody or
Transfer to CMMM or nitrocellulose with lectin
Move. In particular, fractionation of bovine brain cortex homogenate (25 for each)
ug) on a 10% SDS-polyacrylamide gel,
Then at 200 mA in Tris-glycerin buffer for 2 hours
Electricity to either CMMM or nitrocellulose
It migrated electrophoretically. After quenching with BSA,
IMs diluted with anti-protein 1 (synapsin) (1:
300) Rabbit serum was overlaid for 1 hour. Next
Wash with¹²⁵I-labeled protein A (10⁶ cpm
All) incubated for 1 hour, then rewashed and auto
Radiographed. Similar operation to murine red blood cells
It was performed using ghost separation (25 ug each)
Filter quenched by Robin and¹²⁵I-la
Bellization concanavalin A (5 × 10 in total)^Fivecpm) as the probe
Used. When using CMMM,
The much greater binding capacity makes these technologies more sensitive
Further improve. Haptens suitable for the property of lectin binding
Tested. For example¹²⁵I-labeled concanavalin
In the case of A's autoradiograph, each IMs is linked.
The combined net radioactivity is above the bottom of the lane from total counts.
It was determined by subtracting the background measured in. This
Signals on the net CMMM of nitrocellulose
It was 1.6 times higher than the one. 100 mM α-methyl glucosi
The first washing in PBS containing
82% of gnolls have 76 of the signal from nitrocellulose
% Was removed. PB containing 100mA α-methyl mannoside
Second wash in S 90% with CMMM, nitrocell
The removal rate increased by 84% with sucrose. In addition, the acetylcholine receptor is a protein block.
It was analyzed by Ting. Electric manufactured by "Torpedo"
 Lithium Dodecyl Sulfate at 4 ℃
The gel was run on a polyacrylamide gel containing gel. This d
Electroblotte from Lectrohortogram to CMMM
And then quench it with hemoglobin
And then¹²⁵With I-labeled α-bungarotoxin
Overlaid. Only the α-subunit of the receptor
Bound to toxin. This bond is
Non-radioactive alpha-bungarotoxin and tubo
It can be comparable to curarine. The spirit and scope of invention
The methods and products of the present invention may undergo various changes without departing from them.
It can be further modified. Embodiments Disclosed Here
Is for the purpose of detailing the present invention and is not limited thereto.
It is not fixed. [0083] The present invention has a fixed matrix on its surface.
Cation Charge Modified Microporous Nylon Membrane
From the chromatographic substrate that it has,
The present invention relates to a solid-phase assay system for immobilizing offspring in a solid phase
As the solid phase, a polymer is more efficiently used.
It has a large adsorption capacity for immobilization and good retention.
By using a new fixed nylon matrix
Can provide an excellent solid-phase assay system
Became.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 ¹²⁵ I-labeled B for membranes used in the present invention.
It is explanatory drawing which shows the coupling | bonding of SA. FIG. 1a shows the effect of different BSA concentrations on the binding of ¹²⁵ I-labeled protein to IMs. FIG. 1b shows the effect of different concentrations of bovine hemoglobin on ¹²⁵ I-labeled proteins for IMs. FIG. 2 ¹²⁵ I-of various variables into the matrix of FIG.
It is the figure which quantified and showed the movement of labeled BSA. FIG. 3 ¹²⁵ I to the matrix stack of FIG.
-Recovery of labeled BSA. FIG. 4 is a comparative diagram of BSA recovery on a layer of the matrix of FIG.

Claims (1)

Claims: (1) In a solid-phase assay system for immobilizing a polymer in a solid phase from a chromatographic substrate having a cationic charge-modified microporous nylon membrane as an immobilization matrix on its surface, An improved system using a cationic charge-modified microporous membrane comprising a hydrophilic membrane having pores throughout the membrane, the surface of which is decorated with a cationic charge modifier. (2) In a solid-phase assay system in which a polymer is immobilized in a solid phase from a chromatographic substrate having a cationic charge-modified microporous nylon membrane as an immobilization matrix on its surface, the solid phase is spread over the entire membrane. A hydrophilic microporous membrane comprising a substantially isotropic, skinless, porous and hydrophilic nylon microporous membrane having a fine structure, wherein the microporous membrane is Comprising the use of a cationic charge modified microporous membrane comprising an effective charge modifying amount of a cationic charge modifier bound to substantially all of the microstructure without substantial pore size reduction or pore destruction. Improved system. (3) The charge modifier is a reaction product of polyamine and epichlorohydrin, and the reaction product is (i) a tertiary amine group or a quaternary ammonium group, and (ii) an epoxide along the polyamine chain. 3. An improved system as claimed in claim 1 or 2 which has groups, the epoxide groups being capable of binding to the membrane. (4) The charge modifier is a reaction product of polyamine and epichlorohydrin, and the reaction product is one polymer.
A chain and having (i) a tertiary amine or quaternary ammonium group along the chain, and (ii) an epoxide group,
An improved system according to claim 1 or 2 wherein said epoxide groups are capable of bonding to this membrane. (5) The charge modifier is a polyamide-polyamine epichlorohydrin resin.
Improved system described in paragraph. (6) The improved system according to claim 1 or 2, wherein the charge modifier is a polyamine-epichlorohydrin resin. (7) The improved system according to claim 1 or 2, wherein the charge modifier is a diallyl nitrogen-containing substance reacted with epichlorohydrin. (8) A part of the epoxide groups on the charge modifier bonded to substantially all of the microstructure of the microporous nylon membrane has the following (i) at least one primary amine or at least two secondary amines. Aliphatic amines that are polyamines having ## STR3 ##; and (ii) bound to a secondary charge modifier selected from the group consisting of at least one secondary amine and aliphatic amines having carboxyl or hydroxyl substituents. An improved method according to claim 4. (9) The secondary charge modifier has the following general formula 1 [In the formula, R ₁ and R ₂ are alkyl groups having 1 to 4 carbon atoms,
x represents an integer of 0 to 4], The improved system according to claim 8, which is an aliphatic amine. (10) The aliphatic amine has the following general formula 2 10. The improved system according to claim 9, which is tetraethylene pentamine represented by (11) The improved method according to claim 1, wherein the film is substantially isotropic and has no skin. (12) The surface of the pores is modified with the charge modifier,
And (i) an aliphatic amine that is a polyamine having at least one primary amine or at least two secondary amines; and (ii) an aliphatic amine having at least one secondary amine and a carboxyl or hydroxyl substituent. The improved system of claim 2 modified with a secondary charge modifier selected from the group consisting of the following classes: (13) The improved system according to claim 12, wherein a part of the epoxide groups in the charge modifier is bonded to the secondary charge modifier. (14) The improved system according to claim 1 or 2, wherein the substrate is an electrophoretic gel used in gel electrophoresis technology. (15) The improved system according to claim 14, wherein the gel is polyacrylamide. (16) The charge modifier has the following (i) aliphatic amines having at least one primary amino or at least two secondary amino groups; and (ii) at least one secondary amino and carboxyl or hydroxyl substitution. Selected from the group consisting of aliphatic amines having a group,
The improved system of claims 1 or 2 and wherein said charge modifier is attached to the microporous membrane structure through an aliphatic polyepoxide crosslinker having a molecular weight of about 500 or less. (17) The improved system according to claim 16, wherein the polyepoxide comprises 1,4-butanediol diglycidyl ether. (18) The improved system according to claim 17, wherein the charge modifier is tetraethylenepentamine. (19) The improved system according to claim 16, wherein the substrate comprises an electrophoretic gel used in gel electrophoresis technology. (20) The improved method system according to claim 1 or 2, wherein the pore size of the membrane is 0.05 to 1.2 µ. (21) The remaining binding sites of the charge-modified microporous membrane are quenched after protein transfer and before ligand incubation to minimize non-specific background. Alternatively, the improved system according to item 2. (22) The charge modifier is a water-soluble organic polymer having a molecular weight of about 1000 or more, and the polymer is along the chain,
The system according to claim 1 or 2, which comprises (i) a polymer having a polymer chain having a tertiary amine or quaternary ammonium group, and (ii) an epoxide group. (23) The cationic charge modifier is a reaction product of a polyamine and epichlorohydrin, and the reaction product has a tertiary amine or a quaternary ammonium group, and the agent has a substantial structure of the membrane microstructure. A system as claimed in any of claims 53 or 54, all coupled together. (24) The secondary charge modifier is (i) an aliphatic amine having at least one primary amine or at least two secondary amine groups; and (ii) at least one secondary amine and carboxyl or hydroxyl substitution. 24. The system of claim 23 selected from the group consisting of aliphatic amines having groups. (25) The secondary charge modifier is (i) an aliphatic amine having at least one primary amine or at least two secondary amine groups; and (ii) at least one secondary amine and carboxyl or hydroxyl substitution. 24. The system of claim 23 selected from the group consisting of aliphatic amines having groups.