JP2006026607A - Adsorbent for affinity chromatography - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an adsorbent capable of selectively removing IgG and known high-content proteins in blood (serum albumin, and the like) besides IgG from a sample containing a protein group which constitutes blood components by a one-time simple operation. <P>SOLUTION: In the adsorbent for affinity chromatography, antibodies against protein A or protein G and the known high-content proteins in blood besides IgG are bound to an insoluble carrier so as to be presented on the surface of the adsorbent. A solution containing blood components is treated by using the adsorbent and, thereby, IgG and the known high-content proteins in blood are simultaneously and selectively separated from a protein mixture and are removed. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  In the present invention, IgG and a known protein other than IgG (for example, serum albumin) contained in a high amount in blood-derived samples such as blood, plasma, serum, or cell culture supernatant containing serum are simultaneously treated in a single treatment. Adsorbent for affinity chromatography to adsorb and separate from other trace proteins, method for simultaneous removal of known high blood protein other than IgG and IgG using the adsorbent, and IgG and IgG comprising the adsorbent The present invention relates to a kit for simultaneous removal of known high blood protein other than the above.

In recent years, various attempts have been made to find a trace substance peculiar to a disease and to use it for diagnosis of the disease by detecting the trace substance. In addition, blood-derived samples such as blood, plasma, and serum are often used to find such trace substances (markers) or to detect trace substances.
However, blood-derived samples as described above generally contain proteins such as serum albumin, IgG, IgA, antitrypsin, transferrin, and haptoglobin at a high content of 2% to 60% of the total protein. The total content reaches 80% to 90%. When detecting and analyzing other proteins that are present in a very small amount in a blood-derived sample, the presence of a known high-content protein in the blood often becomes an obstacle. For example, serum albumin and IgG significantly interfere with detection of other proteins in protein component analysis by two-dimensional gel electrophoresis or mass spectrometry. Therefore, in such an analysis, it is highly desirable to remove in advance serum albumin and IgG, and preferably high blood content proteins such as IgA, antitrypsin, transferrin and haptoglobin in advance. Also, when purifying trace substances from a blood-derived sample, it is preferable to first remove a known protein contained in a high content from the sample in order to efficiently proceed with the subsequent purification process.

  Affinity chromatography is often used as a means for purifying or removing specific proteins. In affinity chromatography, an antibody against a target substance or a substance (ligand) that has an affinity for the target substance is immobilized on an insoluble carrier, and the target substance is adsorbed specifically to this ligand, so that the target substance is removed from the contaminants. To separate. Further, the adsorbed target substance is eluted by lowering the affinity with the ligand, and it is possible to purify it by collecting it.

Protein A is a protein derived from Staphylococcus and has the property of specifically binding to the Fc region of IgG derived from various animals, and is widely used for IgG purification from serum, cell culture supernatant, ascites, etc. It's being used. Protein G is a protein derived from streptococcus (Streptococcus), and specifically binds to IgG derived from a wide range of animals. For this reason, a technique for immobilizing protein A or protein G on an insoluble carrier and specifically separating and purifying IgG by affinity chromatography using this carrier is widely known. [Kronval, G. et al. et al; immunol. 104, p140-147, 1970, Bjorck, L .; et al; immunol. 133, p969-974, 1984]
Cibacron blue is known as a substance that binds serum albumin, and a method for purifying or removing serum albumin by affinity chromatography using this as a ligand is known [Gianazza, E .; et al; Biochem. J. et al. 201, p129-136, 1982].

  According to the prior art, proteins with high contents such as IgG and serum albumin from blood-derived samples such as blood, plasma, serum, or cell culture supernatant containing serum using affinity chromatography techniques. In order to remove the protein, it is necessary to prepare ligands with specific affinity for each protein to be removed, and to remove each protein to be removed one by one using an adsorbent in which each ligand is immobilized on an insoluble simple substance. . For example, in order to remove serum albumin and IgG, which are known proteins having the highest content, from a blood-derived sample, first, using an adsorbent in which protein A or protein G is immobilized on an insoluble carrier, It is conceivable to remove albumin in a sample using an adsorbent in which IgG is removed and Cibacron Blue is immobilized on an insoluble carrier. However, in this method, the adsorption operation must be performed at least twice, and it takes time, so that loss or inactivation occurs during the removal process, and the target trace protein cannot be efficiently detected or recovered. There was a difficulty.

  In order to overcome this difficulty, a method for simultaneously removing serum albumin and IgG has been proposed and put into practical use. For example, Non-Patent Document 1 discloses a method using two types of adsorbents, an adsorbent in which an antiserum albumin antibody is immobilized on an insoluble carrier and an adsorbent in which an anti-IgG antibody is immobilized on an insoluble carrier. A kit containing such an adsorbent is available from Amersham Biosciences under the trade name “Albumin and IgG Removal Kit”. Non-Patent Document 2 describes a method using two types of adsorbents, an adsorbent in which Cibacron Blue is immobilized on an insoluble carrier and an adsorbent in which protein A is immobilized. A kit containing "Aurum Serum Protein Mini Kit" is available from Bio-Rad Laboratories. Furthermore, Patent Document 1 describes a method using two types of adsorbents, an adsorbent comprising a biotinylated antiserum albumin antibody-avidin-matrix and an adsorbent comprising a biotinylated protein A-avidin-matrix. Yes.

  The above three methods are excellent in that serum albumin and IgG can be removed at a high removal rate. However, the method of Non-Patent Document 1 uses two types of antibodies (that is, antiserum albumin) for preparing an adsorbent. Antibody and anti-IgG antibody) had to be prepared. In addition, Cibacron Blue used in the method of Non-Patent Document 2 has a drawback that it lacks specificity because it binds to proteins other than serum albumin, such as blood coagulation factors and interferons. Furthermore, the method of Patent Document 1 not only requires biotinylation of antibody and protein A, but also does not use an insoluble carrier, so these adsorbents are further bound to avidin and serum albumin and IgG are bound. There is a drawback that the operation is complicated, for example, it is necessary to separate the supernatant by centrifugation.

US Pat. No. 6,410,692 Life Science News, Fall 2003 (issued in September), Amersham Biosciences (Amersham Biosciences) Proteomics, 2003, 3, 1980-1987

  An object of the present invention is to provide an adsorbent for affinity chromatography capable of selectively and simultaneously removing IgG and other known high-content proteins other than IgG from a blood-derived sample containing many kinds of proteins by a simple operation. Is to provide. Another object of the present invention is to provide a method for simultaneously removing IgG and a known high-content protein other than IgG using the adsorbent for affinity chromatography. Furthermore, another object of the present invention is to provide an affinity chromatography column packed with the above-mentioned adsorbent for affinity chromatography, or a kit for simultaneous removal of IgG and a known high-content protein other than IgG comprising the adsorbent. Is to provide.

The present inventors have conducted various studies on a method for simultaneously and selectively removing a known high-content protein other than IgG and IgG from a blood-derived sample by a simple operation. As a result, protein A or protein G and Succeeded in creating an adsorbent that was immobilized so that both antibodies against known high-protein proteins in the blood were displayed on the surface of the adsorbent, and that the above-mentioned object could be achieved by using affinity chromatography using this adsorbent. As a result, the present invention has been completed. That is, the present invention
(1) An adsorbent for affinity chromatography, wherein protein A or protein G and an antibody against a known high-content protein other than IgG are bound to an insoluble carrier so as to be displayed on the adsorbent surface;
(2) Protein A or protein G is bound to an insoluble carrier, and an antibody against a known high protein content in blood other than IgG is bound to a part of protein A or protein G bound to the insoluble carrier. The adsorbent for affinity chromatography according to (1),
(3) The adsorbent for affinity chromatography according to the above (1), wherein an antibody against a known high-content protein other than protein A or protein G and IgG is directly bound to an insoluble carrier,
(4) Adsorption for affinity chromatography according to any one of the above (1) to (3), wherein the known high blood protein other than IgG is serum albumin, transferrin, IgA, haptoglobin, antitrypsin, fibrinogen, hemoglobin or keratin. body,
(5) The adsorbent for affinity chromatography according to any one of (1) to (3), wherein the known high blood protein other than IgG is serum albumin,
(6) The adsorbent for affinity chromatography according to any one of (1) to (5), wherein the insoluble carrier is a film.
(7) Contacting the adsorbent for affinity chromatography according to any one of claims 1 to 3 with a blood-derived sample, and simultaneously adsorbing IgG and a known high-content protein other than IgG in the sample to the adsorbent. A method for simultaneous removal of IgG and a known high-content protein other than IgG,
(8) The simultaneous removal method according to (7), wherein the sample is blood, plasma, serum, or a cell culture supernatant containing serum,
(9) The simultaneous removal method according to the above (7), wherein the known high blood protein other than IgG is serum albumin, transferrin, IgA, haptoglobin, antitrypsin, fibrinogen, hemoglobin or keratin,
(10) The simultaneous removal method according to the above (7), wherein the known high blood protein other than IgG is serum albumin,
(11) An affinity chromatography column formed by packing the affinity chromatography adsorbent according to any one of (1) to (6), and (12) the affinity chromatography according to any one of (1). A kit for simultaneous removal of IgG and a known high-content protein other than IgG comprising the adsorbent for use;
About.

  By using the adsorbent of the present invention, IgG and a known protein other than IgG (such as serum albumin) contained in a high content in blood can be simultaneously and selectively removed by a simple operation. This facilitates the detection and purification of trace proteins that constitute blood components. Furthermore, when the known protein other than IgG is only serum albumin (that is, when serum albumin and IgG are removed at the same time), it is not necessary to use two types of antibodies, so that it is easy to prepare an adsorbent. .

  The adsorbent for affinity chromatography of the present invention is an affinity chromatography formed by binding to an insoluble carrier so that antibodies against known high-content proteins other than protein A or protein G and IgG are displayed (exposed) on the adsorbent surface. Adsorbent. As such an adsorbent, (1) a part of protein A or protein G in which protein A or protein G is bound to an insoluble carrier and an antibody against a known high-content protein other than IgG is bound to the insoluble carrier (2) Protein A or Protein G, and an affinity chromatography adsorbent obtained by directly binding an antibody against a known high protein content in blood other than IgG to an insoluble carrier. It is done.

  In the present invention, “known high blood protein” means a known blood protein (IgG, serum albumin, transferrin, IgA, 2% by mass or more based on the total protein originally contained in blood). Haptoglobin, antitrypsin, fibrinogen, etc.), and keratin and hemoglobin that may be mixed in blood-derived samples such as serum due to human factors are also included.

  In addition to protein A or protein G, known blood other than IgG and IgG can be obtained by using antibodies against known high blood content proteins other than IgG (eg, serum albumin, transferrin, IgA, haptoglobin, antitrypsin, fibrinogen, etc.) An adsorbent that simultaneously removes medium to high protein content can be created. For example, as described below, by preparing an adsorbent for affinity chromatography in which protein A or protein G and an antiserum albumin antibody are bound to an insoluble carrier so as to be displayed (exposed) on the adsorbent surface, An adsorbent that simultaneously removes serum albumin and IgG can be prepared. The production of the adsorbent when the antibody is a serum albumin antibody will be described below. The antibody against a known high blood content protein other than IgG is an antibody other than serum albumin antibody (transferrin, IgA, haptoglobin, antitrypsin, fibrinogen, etc. In the case of the antibody against the serum albumin antibody, the target adsorbent can be prepared in the same manner as the serum albumin antibody. In addition, when removing keratin and hemoglobin, which are often mixed into serum or the like due to human factors, it goes without saying that the same purpose can be achieved by using antibodies against them.

(Serum albumin binding ligand)
The anti-serum albumin antibody that can be a constituent of the adsorbent for affinity chromatography of the present invention is not particularly limited, but is preferably an antibody against serum albumin of the same species as the animal species from which serum albumin to be removed from the sample is derived. . For example, when human cells are cultured in a medium containing fetal bovine serum (FBS) and it is desired to analyze proteins contained in this medium, bovine serum albumin and IgG are removed. It is preferable to use an antibody against serum albumin. However, if the antibody has an affinity for the animal species of the sample, it does not necessarily have to be the same animal species.

  The antiserum albumin antibody according to the present invention may be a polyclonal antibody or a monoclonal antibody.

  Such an anti-serum albumin antibody may be an antibody sold by various vendors, and it is known to produce a known antibody using serum albumin derived from the animal species that is the source of the sample or a partial peptide of serum albumin as an antigen. It can also be produced according to a method.

  Specifically, the anti-serum albumin antibody according to the present invention is, for example, “Protein / Enzymology vol. 182 published by by Method of Enzymology vol. It can be produced according to the method described in “ACADEMIC PRESS, INC. 1990”.

(IgG binding ligand)
Examples of the protein A or protein G that is a constituent of the adsorbent for affinity chromatography of the present invention include protein A derived from Staphylococcus or protein G derived from Streptococcus. These are known to bind to the Fc region of IgG of various animal species, and whether to select protein A or protein G can be appropriately selected depending on the animal species from which the IgG to be removed is derived. it can. IgG from humans, dogs, rabbits, and pigs shows high binding to protein A, but IgGs from rats, horses, sheep, goats, etc. have higher binding properties to protein G. The protein A or protein G can be obtained by culturing each of the aforementioned bacteria and isolating and purifying it using a known method. Or it can obtain by the protein expression method by gene recombination. A method for producing protein A or protein G by a gene recombination technique is well established in the technical field, and may be followed by a conventional method.

(Selection of insoluble carrier and binding of protein A or protein G)
Any insoluble carrier that can immobilize protein A or protein G and is insoluble in water may be used as the insoluble carrier used in the affinity chromatography in the present invention. Examples thereof include cellulose, agarose, dextran, polyacrylamide, silica gel, and porous glass.

  As a method for immobilizing an antibody and protein A or protein G as an ligand on an insoluble carrier, a known method may be used. For example, a method of binding a gel and a ligand via an amino group, a carboxyl group, a thiol group, a hydroxyl group or the like present in the ligand is well known. Bromocyan, bisepoxide, epichlorohydrin, N-hydroxysuccinimide and the like are typical crosslinking agents. As an example, agarose can be treated with a cross-linking agent (eg, bromocyan) to bind the amino group of the ligand.

  A spacer can be interposed between the insoluble carrier and the ligand. Insoluble carriers treated with the above-mentioned crosslinking agent and added with spacers are commercially available from various companies, and the use of them can simplify the operation of ligand immobilization. The procedure for ligand immobilization varies depending on the crosslinking agent used.

Insoluble carriers (including those treated with a crosslinking agent) used in the present invention are commercially available from various companies. For example, Sepharose 2B, Sepharose 4B, Sepharose 6B, CNBr manufactured by Amersham Biosciences are listed as examples of commercially available products. -Activated Sepharose 4B, AH-Sepharose, CH-Sepharose, Epoxy-Activated Sepharose 6B, etc. Bio-Gel A, Cellex, Aminoethyl-Bio-Gel, Bio-Gel CM, Affi- manufactured by Bio-Rad Laboratories Examples thereof include Gel15, Affi-Pre10, Chromagel A, Chromagel P, Enzafix P-HZ, etc. manufactured by Wako Pure Chemical Industries, Ltd., AE-cellulose, CM-cellulose, PAB cellulose, etc. manufactured by SELVA.
Moreover, the shape of the insoluble carrier (including those treated with a crosslinking agent) used in the present invention is not particularly limited, and may be granular or film-like.

  As a procedure for binding each ligand to an insoluble carrier, it is possible to react protein A or protein G and an antibody with an insoluble carrier treated with various crosslinking agents at the same time. Is reacted with an insoluble carrier treated with a cross-linking agent and immobilized. Thereafter, a procedure is used in which the antibody is allowed to act on immobilized protein A or protein G to be immobilized. At this time, since protein A or protein G and the Fc region of the antibody specifically bind, loss of the antigen binding activity of the antibody can be suppressed to a low level. Further, in order to prevent leakage of the antibody from protein A, after binding protein A or protein G and the antibody, both may be chemically crosslinked with a bivalent crosslinking reagent such as dimethylpimelimidate.

An insoluble carrier on which protein A is immobilized is also commercially available, for example, commercially available from Amersham Biosciences under the trade name “rProtein A Fast Flow Gel”. Furthermore, there are also commercially available anti-serum albumin antibodies, for example, commercially available under the trade name “anti-human albumin” from the Institute of Medical Biology. In the present invention, a commercially available antiserum albumin antibody can be bound to such a commercially available insoluble carrier by the above method.

  The serum albumin binding ability and IgG binding ability of the adsorbent of the present invention also vary depending on the amount and ratio of protein A or protein G and antiserum albumin antibody presented (exposed) on the adsorbent surface. Therefore, in order to efficiently remove serum albumin and IgG in the sample, the amount and ratio of the ligand may be appropriately selected according to the type of sample. For example, when the above-mentioned commercially available r Protein A Fast Flow gel (protein A binding amount: 6 mg protein A / ml swelling gel) is used as an insoluble carrier on which protein A, which is a raw material for producing the adsorbent, is immobilized, swelling It is preferable to use 2 to 4 mg of affinity-purified antiserum albumin antibody, more preferably 2 to 3 mg, most preferably 2.4 to 2.8 mg per 0.1 ml of gel. If the antiserum albumin antibody is used in excess, most of the protein A bound to the insoluble carrier binds to the antibody and loses the IgG binding ability, which is not preferable. Also, if the amount of the antiserum albumin antibody is too small, the serum albumin binding ability becomes weak, which is also not preferable. Usually, the ratio (molar ratio) of protein A or protein G and antiserum albumin antibody presented on the surface of the adsorbent is most preferably 1: 1 to 3: 4.

  The adsorbent of the present invention thus obtained, that is, an adsorbent in which protein A or protein G is bound to an insoluble carrier and the antiserum albumin antibody is bound to a part of protein A or protein G bound to the insoluble carrier. This is schematically shown in FIG. 1-a.

  On the other hand, the adsorbent formed by directly binding protein A or protein G and antiserum albumin antibody to an insoluble carrier first binds the antiserum albumin antibody to an insoluble carrier (including those treated with a crosslinking agent) It can be produced by binding protein A or protein G to the insoluble carrier. Alternatively, the reverse procedure may be used. It is also possible to bind each ligand in an appropriate ratio and simultaneously bind to the insoluble carrier. The preferred ratio of each ligand in this case follows the above. The adsorbent thus obtained is schematically shown in FIG.

  The adsorbent for affinity chromatography of the present invention is an adsorbent in which only protein A or protein G is bound to an insoluble carrier (FIG. 1-c), and an adsorbent in which only an antibody against a known high blood protein other than IgG is bound. The adsorbent (FIG. 1-e) bound to an insoluble carrier may be mixed so that only antibodies against known blood proteins other than IgG are present on the adsorbent surface. Absent.

(Storage conditions)
It is desirable to store the adsorbent on which an antibody against a known high-content protein other than IgG and protein A (or protein G) is immobilized at 4 to 8 ° C. in a buffer solution to which an appropriate bacteriostatic agent is added. The buffer varies depending on the sample used, but when human serum is used, it is preferably a 20 mM phosphate buffer (pH 7.4) containing 150 mM sodium chloride (hereinafter abbreviated as PBS). Examples of the bacteriostatic agent include sodium azide and marthiolate. As an example of storage, it is preferable to store in PBS containing 0.05% sodium azide. Prior to reacting the adsorbent with the sample, the adsorbent gel may be replaced with an appropriate buffer, or may be used as it is.

  Another aspect of the present invention comprises contacting the sample with the adsorbent for affinity chromatography obtained above, and simultaneously removing IgG contained in the sample and a known high blood content protein other than IgG. This is a method for simultaneous removal of IgG and other known high-content proteins other than IgG.

(Sample selection and quantity)
IgG to be removed and known high blood protein content other than IgG are typically present in samples with other trace proteins and / or biomolecules. Examples of the sample include blood and processed / purified products derived from blood (including plasma and serum), cells cultured in a cell culture solution containing serum, and other solutions containing blood components.

  The amount of IgG and high blood content proteins other than IgG that can be removed at once by the method of the present invention is the method of preparing the adsorbent, or the type and amount of the sample used, and the amount of known high blood protein other than IgG and IgG. It depends on the content rate. Moreover, depending on the sample used, dilution may be required. For example, when removing IgG and serum albumin from normal human serum, it is preferable to dilute to about 2 to 5 times with an appropriate buffer (for example, PBS) before use in the method of the present invention. At this time, it is desirable to remove the buffer solution from the adsorbent preservation solution prepared by the above procedure as much as possible and then add it to the diluted sample. In addition, the sample may be added to the adsorbent preservation solution without diluting without removing the buffer solution. Also in this case, when the sample is normal human serum, the ratio of the sample to the buffer is preferably 1: 2 to 5.

(Column selection)
As a chromatographic technique, the sample solution is brought into contact with the adsorbent by a batch method, and IgG and high-content proteins other than IgG contained in the sample are adsorbed, and then a solution containing non-adsorbed components is centrifuged. It collects by the method of. When the adsorbent prepared by the above method is in the form of gel, it is preferable to use it after filling the column. Examples of the column material include glass, plastic, and stainless steel, but glass or plastic is preferable. Although there is no restriction | limiting in particular about the capacity | capacitance of a column, It is desirable to select according to the quantity of the adsorbent to be used. When processing a small amount of sample, it is preferable to use a spin column that can be set in a microtube. After mixing the sample with the adsorbent, it is preferable to leave about 10 to 30 minutes before separating the two in the column. During this time, it is more preferable to shake and mix this mixture about once every 5 minutes. Most preferably, shaking is continued after mixing until separation.

  After separating the solution containing the non-adsorbing component from the mixture of the sample and the adsorbent, it is preferable to use the separated solution together with the flow-through solution after further bringing the adsorbent into contact with an appropriate buffer. It is desirable that the buffer used here has the same composition as that used for dilution of the sample.

(Use of treated sample)
In the affinity chromatography of a blood-derived sample with an adsorbent prepared by the above method, both IgG and a known high blood protein content other than IgG are removed from the sample solution that does not bind to the adsorbent and passes through. By washing the adsorbent with a buffer solution having the same volume as the used adsorbent, the non-adsorbed component can be efficiently recovered. The recovered material after chromatography can be subjected to electrophoresis including, for example, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis. Chromatography of the blood-derived sample using the above adsorbent reduces the concentration of the entire protein in the sample solution, but removes IgG and the content of other proteins other than the known high blood protein other than IgG hardly change. For this reason, if a sample solution containing the same weight of protein is subjected to electrophoresis, a much smaller amount of protein can be detected. It is also possible to concentrate by precipitating the protein of the treated sample with 10% trichloroacetic acid or 5 volumes of cold acetone.

Further, the chromatography of the sample by the adsorbent can be positioned as a means for purifying a trace amount of protein. For example, a trace amount of protein can be purified by subjecting the recovered product after chromatography to an ion exchange chromatography or gel filtration chromatography as it is, or after dilution or substitution with an appropriate buffer.
Furthermore, the use of the adsorbent is also useful for clinical tests using blood samples. By removing IgG and any known high-content protein other than IgG, it becomes easier to observe the trend of trace proteins.

  Yet another embodiment of the present invention is IgG and a kit for removing high-content protein other than IgG, which comprises the adsorbent for affinity chromatography obtained above. In general, the kit may contain, in addition to the adsorbent and the column, a buffer solution for equilibrating the adsorbent, a sample diluent, and the like. Examples of the column include glass, plastic, and stainless steel. As a buffer solution, although it changes also with the sample to apply, PBS is mentioned suitably, for example.

  Examples of the present invention will be described below, but the present invention is not limited thereto. For example, in order to adjust the serum albumin binding ability and IgG binding ability of an adsorbent, the adsorbent of the present invention is an adsorbent or adsorbent in which only protein A (or protein G) is presented (exposed) on the adsorbent surface. It can also be used by mixing with an adsorbent in which only serum albumin antibody is presented (exposed) on the body surface.

(Preparation method of anti-serum albumin antibody-binding protein A gel and treatment conditions of human serum)
[1-1: Examination of ratio of protein A gel to antiserum albumin antibody]
Anti-serum albumin antibody (anti-human albumin rabbit immunoglobulin G solution [Medical Biology Co., Ltd.]) against 50 μl of rProtein A Fast Flow gel (Amersham Biosciences, hereinafter referred to as Protein A gel) previously substituted with PBS. Laboratory], concentration: 29.8 mg / ml, titer: 4.5 mg / ml; hereinafter referred to as anti-albumin antibody solution), 30, 40, 50, and 60 μl each were applied and allowed to react at room temperature for 1 hour. Thereafter, the gel and the solution were separated by centrifugation (250 × g, 3 minutes), and a part of the supernatant was subjected to SDS-PAGE (SDS polyacrylamide gel electrophoresis). As a result, when 50 μl or more of the anti-albumin antibody solution was applied to 50 μl of protein A gel, many unreacted antibodies were detected (FIG. 2). From this, it was judged that the amount of an appropriate anti-albumin antibody solution was 40 μl per 50 μl of protein A gel.

[1-2: Examination of cross-linking reagent concentration]
Protein A gel and anti-albumin antibody solution were mixed at a volume ratio of 5 to 4, and stirred at room temperature for 1 hour, then 0.15, 1.5, 15, 75, 150, 300, 450 mM DMP (dimethyl) (Pimelimidate) solution (0.2 M triethanolamine, pH 8.3) was added in an amount equal to that of protein A gel, and the mixture was stirred at room temperature for 1 hour to cause a crosslinking reaction. A part of the antibody-binding protein A gel cross-linked at each DMP concentration was added to a sample buffer for electrophoresis and heated at 95 ° C. for 5 minutes, and then the supernatant was subjected to SDS-PAGE. As a result, many uncrosslinked antibodies were detected from the supernatant of the antibody-bound protein A gel slurry crosslinked with 15 mM or less of DMP (FIG. 3). From this result, it was found that the crosslinking reaction was incomplete with 15 mM or less of DMP, and 75 mM or more of DMP was required for a more complete crosslinking reaction.

  In addition, 200 μl of antibody-bound protein A gel cross-linked with each concentration of DMP was taken, washed with a 30-fold amount of a washing solution (0.1 M glycine, pH 2.8), and further washed with a 30-fold amount of PBS. 50 μl of PBS and 15 μl of human serum were added to the antibody-binding protein A gel from which the solution had been completely removed, and allowed to stand at room temperature for 30 minutes (stirred every 5 minutes during this time). Thereafter, the gel slurry was transferred to a microbiospin column, and the non-adsorbed fraction was collected by centrifugation (250 × g, 3 minutes). Furthermore, 200 μl of PBS is added to the column to wash the gel, and the washing solution is collected by centrifugation and mixed with the first non-adsorbed fraction that has flowed out. A part of this mixture solution is subjected to SDS-PAGE. (FIG. 4). From this result, it was found that antibody albumin protein A gel cross-linked at a DMP concentration of 150 mM or less did not sufficiently remove serum albumin. From this, it was determined that 300 mM was suitable as the DMP concentration used for the cross-linking reaction. did.

[1-3: Examination of washing conditions for uncrosslinked antibody]
The antibody-bound protein A gel cross-linked with 300 mM DMP was washed with 10 times the washing solution, and this was repeated 10 times. A part of the washing solution for each washing number was subjected to SDS-PAGE, and the outflow state of the uncrosslinked antibody was confirmed (FIG. 5). From this result, it was found that the uncrosslinked antibody could be sufficiently removed by washing 5 times or more, and it was judged that the uncrosslinked antibody could be removed by washing with 50 times the washing solution of the crosslinked antibody-binding protein A gel. .

[1-4: Examination of treatment conditions using human serum samples]
To the tube with lid, 200 μl of antibody-binding protein A gel and 50 μl of PBS were added, and further 10, 15, and 20 μl of human serum were added. These tubes were left at room temperature for 30 minutes (stirred every 5 minutes during this time). Thereafter, the gel slurry in the tube was transferred to a microbiospin column, and the solution was recovered by centrifugation (recovered solution 1). Furthermore, 200 μl of PBS was added to each antibody-binding protein A gel, and the solution was recovered by centrifugation (recovered solution 2) to obtain a column-treated sample (recovered solution 1 + 2). As a result of subjecting a part of each column-treated sample to SDS-PAGE, a large amount of residual serum albumin was observed when 20 μl of serum was applied (FIG. 6). From this, it was judged that the amount of human serum applicable to 200 μl of antibody-binding protein A was 15 μl or less.

As a result of the above examination, an optimal method for producing an anti-serum albumin antibody-binding protein A gel was determined as follows.
First, 160 μl of an anti-albumin antibody solution was applied to 200 μl of protein A gel, and the mixture was stirred at room temperature for 1 hour. Thereafter, an equivalent amount of 300 mM DMP solution to the gel was added to the gel, and the mixture was stirred at room temperature for 1 hour. Next, the gel after DMP crosslinking was washed 10 times with a 10-fold amount of washing solution. After further washing three times with PBS containing 10 times the amount of 0.05% sodium azide, PBS containing 0.25 times the amount of 0.05% sodium azide was added. 250 μl of the thus prepared antiserum albumin-binding protein A gel slurry was taken and transferred to a tube with a lid. To this was added 15 μl of human serum and these tubes were allowed to stand at room temperature for 30 minutes (stirred every 5 minutes during this time). Thereafter, the gel slurry in the tube was transferred to a microbiospin column, and the solution was recovered by centrifugation (recovered solution 1). Furthermore, 200 μl of PBS was added to the antibody-bound protein A gel after serum treatment, and the solution was recovered by centrifugation (recovered solution 2) to obtain a sample after column treatment (recovered solution 1 + 2).
In the following examples, samples were processed using the gel prepared by the above procedure.

(Comparison of serum albumin and IgG removal efficiencies with methods using Cibacron Blue and Protein A)
[2-1: Protein quantification]
Using the gel of the present invention prepared in accordance with the procedure determined in Example 1 (hereinafter referred to as the gel of the present invention), human serum was processed according to the operating procedure determined in Example 1, and included in the sample before and after the treatment. The total protein content was quantified. The operation was performed by measuring the absorbance at 562 nm with a spectrophotometer (U-best 30; JASCO) according to the instructions of BCA Protein Assay Kit (Pierce). As a result, the total protein amount was 1087 μg in 15 μl of human serum before treatment, and 240.8 ± 34.1 μg in 265 μl of serum sample after treatment (n = 4). That is, by using the gel of the present invention, it was found that the total amount of protein in the serum sample was reduced to 22.2 ± 3.1% (77.8% removal rate).

  As a conventional technique, an affinity chromatography kit in which Cibacron Blue F3GA-immobilized agarose gel and protein A-immobilized agarose gel are mixed is commercially available (BIO-RAD "Aurum Serum Protein Mini Kit" Non-Patent Document 2). Using this kit, normal human serum was treated according to the operation method attached to the kit (hereinafter, this treatment is referred to as treatment with Cibacron Blue kit).

Furthermore, the total amount of protein contained in the solution collected from this kit was quantified as described above. As a result, the total protein amount was 3622 μg in 50 μl of human serum before treatment, and it was 942.4 μg in 400 μl of serum sample after treatment with the kit using Cibacron Blue.
That is, it was found that the total protein amount in the serum sample was reduced to 26.0% (removal rate was 74.0%) by the treatment using the Cibacron Blue kit. This was a value slightly lower than the removal rate when the gel of the present invention was used.

[2-2: SDS-PAGE]
A part of each of the eluate collected from the gel of the present invention and the kit using Cibacron Blue and normal human serum before treatment were taken and subjected to SDS-PAGE (FIG. 7).

  As a result, the treatment with the Cibacron Blue kit (lane labeled Cibacron Blue in FIG. 7) is more serum than the treatment with the gel of the present invention (lane labeled antiserum albumin antibody in FIG. 7). It was found that both albumin and IgG had a large amount of elution into the flow-through fraction, and the removal efficiency was poor. Furthermore, it was also confirmed that the band located at a molecular weight of 116 kDa disappeared in the sample after treatment with the kit using Cibacron Blue.

[2-3 Two-dimensional electrophoresis]
Furthermore, each column-treated sample was subjected to two-dimensional electrophoresis, and Sypro Ruby (Molecular Probes) staining was performed. For the electrophoresis, normal human serum before treatment or a collected solution after each column treatment, each having a protein amount of 5 μg was used. Each sample was subjected to lysis buffer (9M UREA, 2% Triton X-100, 2% 2-mercaptoethanol, 0.72% pharmalyte (pH 3 to 10), 20 mM phenylmethylsulfonyl fluoride) containing 50 mM DTT (Dithiothreitol). After denaturation and dissolution, dilute to 100 μl with sample buffer [8M UREA, 0.5% Triton X-100, 2% 2-mercaptoethanol, 0.72% pharmalyte (pH 3-10; Amersham Bioscience)]. Were subjected to electrophoresis. The first dimension was ReadyStrip IPG Strips pH 3-10, 7 cm (BIO-RAD), and the second dimension was 12.5% SDS polyacrylamide gel.

  FIG. 8 is a photographic drawing of two-dimensional electrophoresis of normal human serum (5 μg protein), FIG. 9 is a photographic drawing of two-dimensional electrophoresis after treatment with the gel of the present invention (5 μg protein), and FIG. 2 is a photographic drawing of two-dimensional electrophoresis after treatment with a Clone Blue kit (5 μg protein). In these drawings, a portion containing a serum albumin spot and a partial spot of IgG H chain is surrounded by a thin frame and a partially enlarged image is also mounted. The circled portion in FIG. 9 is a spot that could not be detected during pre-chromatography human serum sample migration, and the portion surrounded by a thick square in FIG. 10 is the spot position that could be detected during pre-chromatography human serum sample migration. Show. Regarding the positions of serum albumin and IgG spots shown in the figure, the SWISS-2DPAGE database (URL / http: //kr.expasy.org/ch2d/) was referred to.

From these results, it can be seen that the serum albumin and IgG spots are significantly reduced by the treatment with the gel of the present invention, and thus the amount of electrophoretic protein can be increased, and it is close to the serum albumin and IgG present in the serum. It was confirmed that it was easy to detect the spot at the position. In addition, no spots where the staining intensity decreased were observed.
On the other hand, in the treatment with Cibacron Blue kit, in addition to the poor removal efficiency of serum albumin and IgG as compared with the treatment with the gel of the present invention, a plurality of spots in which the staining intensity decreased or disappeared were confirmed.

  When these spot proteins were estimated from the SWISS-2DPAGE database, IgD, IgM, Hemopexin, ApoAI, and ApoD were listed as spots that could be detected or increased by the gel of the present invention. Many other unidentified spots were also detected. On the other hand, the spots decreased by the treatment using the Cibacron Blue kit are plasminogen, ApoJ, ApoE, and α2-Antiplasmin. Furthermore, a plurality of unidentified spots were reduced or disappeared.

(PMF (peptide mass fingerprinting) analysis)
Using the gel of the present invention, after treatment and pretreatment serum (protein amount 1.5 μg) were subjected to reductive alkylation treatment, each solution was enzymatically digested with 0.5 μg trypsin (35 ° C., left overnight). And desalted and concentrated, and then PMF analysis was performed using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (Voyager-DE STR: Applied Biosystems) (FIG. 11).

  As a result, since the amount of serum albumin in human serum before treatment is large, the signal of serum albumin accounts for 17 (85%) in the top 20 detected peptide signals, and 3 signals other than serum albumin (15%) is detected, whereas serum after treatment with the gel of the present invention reduces the serum albumin signal to 7 (35%) and 13 signals other than serum albumin (65%) ). Thus, it was confirmed that proteins other than serum albumin and IgG can be easily detected in PMF analysis by treating serum using the gel of the present invention.

  By using the adsorbent for affinity chromatography of the present invention, IgG and known proteins other than IgG contained in a high content in blood can be removed simultaneously and selectively by a simple operation. It is possible to easily detect and purify the constituent trace protein.

1- (a) and (b) are schematic views of the adsorbent according to the present invention, and FIGS. 1- (c), (d) and (e) are mixed in the adsorbent according to the present invention. It is a schematic diagram of a good adsorbent. It is the result of examining the liquid volume of the anti-albumin antibody solution made to react with protein A, and is a photographic drawing when the antibody protein is detected by SDS-PAGE of the post-reaction supernatant and CBB staining. It is a result of examining the DMP concentration in the crosslinking reaction between protein A and antibody, and is a photographic drawing when the amount of uncrosslinked antibody contained in the gel slurry after the reaction is compared by SDS-PAGE. It is a result of examining the DMP concentration in the crosslinking reaction between protein A and an antibody, and is a photographic drawing when the serum albumin and IgG removing ability of the antibody-bound protein A gel after the crosslinking reaction is compared by SDS-PAGE. It is the result of examining the washing conditions of uncrosslinked antibody from antibody-bound protein A gel after crosslinking reaction. A washing solution was collected for each washing after the crosslinking reaction, and the amount of uncrosslinked antibody contained in each washing solution was determined by SDS-PAGE. FIG. It is a result of examining the amount of human serum that can be treated with 200 μl of the gel of the present invention, and is a photographic drawing when the amounts of serum albumin and IgG remaining in the collected sample after treatment are compared by SDS-PAGE. . It is a photograph drawing when the processing result by the column of a prior art and the processing result by the gel of this invention are compared by SDS-PAGE. 2 is a photographic drawing of two-dimensional electrophoresis of normal human serum (5 μg protein). 2 is a photographic drawing of two-dimensional electrophoresis after treatment with the gel of the present invention (5 μg protein). It is a photograph drawing of the two-dimensional electrophoresis of the process liquid (5 microgram protein) by a Cibacron blue use kit. It is a figure which shows the result of carrying out the enzyme digestion after the reductive alkylation and the PMF analysis of the solution (b) which processed human serum with the gel of this invention, and the unprocessed solution (a).

Explanation of symbols

1 Insoluble carrier 2 Protein A or Protein G
3 Anti-serum albumin antibody M Lane of molecular weight marker (broad range) of Aproscience A Lane in which the reaction solution after the crosslinking reaction was run

Claims (12)

  An adsorbent for affinity chromatography, wherein protein A or protein G and an antibody against a known high-content protein other than IgG are bound to an insoluble carrier so as to be displayed on the adsorbent surface.   Protein A or protein G is bound to an insoluble carrier, and an antibody against a known high-content protein other than IgG is bound to a part of protein A or protein G bound to the insoluble carrier. Item 4. The adsorbent for affinity chromatography according to Item 1.   The adsorbent for affinity chromatography according to claim 1, wherein an antibody against a known high protein content in blood other than protein A or protein G and IgG is directly bound to an insoluble carrier.   The adsorbent for affinity chromatography according to any one of claims 1 to 3, wherein the known high protein content in blood other than IgG is serum albumin, transferrin, IgA, haptoglobin, antitrypsin, fibrinogen, hemoglobin or keratin.   The adsorbent for affinity chromatography according to any one of claims 1 to 3, wherein the known high blood protein other than IgG is serum albumin.   The adsorbent for affinity chromatography according to any one of claims 1 to 5, wherein the insoluble carrier is a film.   The adsorbent for affinity chromatography according to any one of claims 1 to 3 is brought into contact with a blood-derived sample, and IgG and high blood content protein other than IgG in the sample are simultaneously adsorbed to the adsorbent. A method for simultaneously removing IgG and a known high-content protein other than IgG.   The simultaneous removal method according to claim 7, wherein the sample is blood, plasma, serum, or a cell culture supernatant containing serum.   8. The simultaneous removal method according to claim 7, wherein the known high blood protein other than IgG is serum albumin, transferrin, IgA, haptoglobin, antitrypsin, fibrinogen, hemoglobin or keratin.   The simultaneous removal method according to claim 7, wherein the known high blood protein other than IgG is serum albumin.   An affinity chromatography column formed by packing the affinity chromatography adsorbent according to claim 1. A kit for simultaneous removal of IgG and a known high-content protein other than IgG, comprising the adsorbent for affinity chromatography according to any one of claims 1 to 6.