JP5089924B2 - Method for purifying IgM type antibody, adsorbent for IgM type antibody recognition antigen - Google Patents

Description

  The present invention relates to a novel method for purifying IgM-type antibodies having higher purity and higher activity than those in a sample at a low cost, or an adsorbent for IgM-type antibody recognition antigen.

  Antibodies, or immunoglobulins, are classified into the classes IgM, IgG, IgA, IgE, and IgD. Among them, the IgM type antibody has the largest molecular weight, and generally exists as a pentamer composed of 10 heavy chains and 10 light chains. IgM antibodies are the earliest antibodies produced in the immune response and are thought to play a major role in protecting against bacterial infections. IgM antibodies have a lower affinity for antigens than IgG antibodies, which are the main immunoglobulins in blood, but they can bind to antigens at multiple points and can efficiently stimulate the complement system of animals. The ability as is expected. In addition, since it has high specificity for polysaccharides, it has been attempted to apply it to cancer diagnosis as an immunoglobulin specific to a cancer-related sugar chain. For these reasons, the utility value of IgM antibodies in the medical field is extremely high today.

  The most common method for producing a monoclonal IgM antibody is a cell fusion method using hybridoma cells (G. Kohler, C. Milstein, Nature 256, 495, 1975). A hybridoma cell is a cell prepared by synthesizing a monoclonal IgM antibody by fusing a B cell having antibody-producing ability and a myeloma cell having proliferative ability. As a method for obtaining an IgM type antibody solution from a hybridoma cell, a method of obtaining a solution containing a monoclonal IgM type antibody by culturing the hybridoma cell in a serum medium and a serum-free medium, etc. A method for obtaining a solution containing a monoclonal IgM antibody from the ascites can be considered. Other methods for preparing an IgM type antibody include a method for obtaining a monoclonal IgM type antibody from B cells derived from a myeloma patient and a method for obtaining it from the blood of a macroglobulinemia patient.

  However, since blood and culture solution containing these IgM antibodies contain proteins, lipids, and components derived from the cell culture solution other than the target IgM antibodies, the impurities are the purity of the purified IgM antibodies. The effect on the recovery rate is considered to be extremely large. In particular, serum and plasma components account for a high proportion of low-density lipoprotein (LDL) and other contaminating proteins, making it extremely difficult to obtain highly pure IgM antibodies.

  In the purification of IgG type antibodies, the use of protein A or protein G immobilization materials has become a common technique, but this has low affinity for IgM type antibodies and is not suitable for purification of IgM type antibodies. . Further, since the binding between the antibody and protein A or protein G is very strong, it is necessary to use a strongly acidic solution of about pH 3 to separate and recover the bound antibody. For this reason, there is a problem that it is difficult to recover in a state where the original antibody activity is retained. Furthermore, protein A and protein G cannot be used depending on the animal species of the antibody, and since protein A and protein G are produced from fungi, mass production is difficult, and materials for purifying the antibody are very expensive. There is also the problem of being. In addition, since the material is derived from fungi, there is concern about the introduction of toxic substances, and safety problems cannot be completely eliminated.

  Regarding the purification of IgM-type antibodies, affinity chromatography such as a material on which anti-IgM antibodies are immobilized has been developed, but the same problems as described above are considered. There are various other purification methods, but purification alone is not sufficient, and it is necessary to combine a plurality of purification methods, and there is a problem that purification requires complicated and long-time operation. Therefore, development of a simple, highly pure and highly active IgM-type antibody purification method is desired.

  An object of the present invention is to provide a novel method for easily purifying a highly pure and highly active IgM antibody from a sample, or an antigen adsorbent recognized by an IgM antibody.

  The present inventors diligently studied a method for simply purifying a highly pure and highly active IgM antibody from an IgM antibody sample containing contaminating components. As a result, the IgM-type antibody-containing solution was pretreated to remove substances other than the IgM-type antibody having high affinity for the sodium urate crystals, and then contacted with the sodium urate crystals to selectively convert the IgM-type antibodies to the sodium urate crystals. It was found that IgM type antibodies having high purity and high activity can be easily purified by binding, and the present invention was completed.

  That is, the present invention relates to a method for efficiently purifying a highly pure and highly active IgM type antibody.

  The pretreatment in the present invention is to bring an IgM-type antibody-containing solution into contact with an adsorbent having a cation exchange ability. Specifically, the adsorbent having a cation exchange ability immobilizes an anionic compound such as dextran sulfate. It is an adsorbent formed.

  Further, as a pretreatment, the IgM antibody-containing liquid may be brought into contact with an adsorbent having an anion exchange ability, or an adsorbent having a cation exchange ability and an adsorbent having an anion exchange ability may be used in combination.

  The method for purifying IgM-type antibodies of the present invention makes it possible to efficiently and simply collect and purify IgM-type antibodies that are expected to be applied to therapeutic agents and diagnostic agents. Type antibodies can retain high activity. In addition, it is not necessary to use affinity chromatography called Protein A or Protein G having high affinity for the antibody, and it is possible to separate and purify at low cost.

  The pretreatment in the present invention is to adsorb and remove substances other than the IgM-type antibody having high affinity for sodium urate crystals by bringing the IgM-type antibody-containing liquid into contact with an adsorbent having a cation exchange ability.

  The adsorbent having cation exchange capacity is an adsorbent obtained by immobilizing an anionic compound such as dextran sulfate.

  The adsorbent is preferably a water-insoluble carrier that is solid at normal temperature and pressure. Water-insoluble carriers include granular, plate-like, fiber-like, and hollow fiber-like shapes, but the shape is not particularly limited. For example, organic beads composed of inorganic carriers such as glass beads and silica gel, synthetic polymers such as crosslinked polyvinyl alcohol, crosslinked polyacrylate, crosslinked polyacrylamide, and crosslinked polystyrene, and polysaccharides such as crystalline cellulose, crosslinked cellulose, crosslinked agarose, and crosslinked dextrin Representative examples include carriers, and composite carriers such as organic-organic and organic-inorganic obtained by combinations thereof.

  The IgM-type antibody-containing liquid in the present invention includes blood, plasma, serum, ascites, lymph, intra-articular fluid, fractional components obtained therefrom, and other liquid components derived from living organisms, or in vitro cell culture fluid. Although Kiyoshi etc. can be considered, it is not particularly limited to these.

  IgM-type antibody-containing liquids, for example, blood-derived liquid components such as blood and cell culture fluids contain many contaminating components in addition to lipid / protein components such as LDL, components derived from cell culture fluids, etc. . Therefore, it is very difficult to purify and separate highly pure IgM type antibodies. Since the pretreatment according to the present invention can adsorb and remove substances other than the IgM type antibody having a high affinity for the sodium urate crystals, the IgM type antibody can be selectively bound to the sodium urate crystals to obtain a high purity IgM type antibody. It is possible to obtain.

  As a specific method of use, for example, a highly pure and highly active IgM type antibody can be purified from an IgM type antibody-containing solution containing contaminating components by the following procedure, but the present invention is not limited to the following specific examples. . That is, as a pretreatment, an adsorption type plasma purifier liposorber (Kaneka Co., Ltd.) having a high adsorption capacity for LDL, VLDL, and Lp (a) is packed in the column, and an IgM type antibody-containing solution is passed through the column. By adsorbing and removing impurities. Then, the column was filled with sodium urate crystals prepared according to the method of McCarty (McCarty) and Faires (Curr Ther Res 5, Vol. 284, 1963). An IgM-type antibody-containing solution excluding contaminating components is passed through to selectively bind the IgM-type antibody to sodium urate crystals. Subsequently, the remaining contaminating components are washed with an appropriate solution, and then the IgM type antibody bound to the sodium urate crystal is eluted to recover the highly pure and highly active IgM type antibody.

  The sodium urate crystals to which the IgM antibody bound by the present invention is bound can be applied to an affinity column that adsorbs the antigen recognized by the IgM antibody.

  In addition, since the IgM antibody obtained by the method of the present invention has high activity, it can be applied to an affinity column that is immobilized on another carrier and adsorbs an antigen recognized by the IgM antibody.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited only to a following example.

Example 1
Whole serum was passed through an LDL removal column packed with Liposorber (manufactured by Kaneka Corporation) to obtain an LDL-removed fraction and an LDL fraction eluted with a 1M sodium chloride solution. The LDL-removed fraction was further passed through a microgranular cellulose anion exchange column DE52 (Whatman) to obtain an IgG fraction and an IgG-removed fraction. Further, Zymosan was added to the LDL-removed fraction and incubated at 37 ° C. for 30 minutes to obtain a complement consumption fraction.

  Total serum or each serum fraction is added to sodium urate crystals prepared by the method of McCarty & Faires, incubated at 37 ° C. for 30 minutes, washed several times with sodium urate saturated physiological saline, and then limited molecular weight of 6-8000. A sodium urate adsorbed protein was obtained by placing in an outer filtration membrane (Spectra / Por 1) and dialyzing with pure water until sodium urate was completely dissolved. These operation steps are shown in FIG.

  SDS-PAGE of the serum and serum fractions obtained by the above-described method and the respective sodium urate adsorbed protein was performed and CBB staining was performed, and the results shown in FIG. 2 were obtained. For the sodium urate adsorbed protein from the whole serum and LDL-removed fractions, cut out the band that was highly stained by CBB staining, digest it in gel with trypsin, analyze it by LC-MS, and identify the protein from the obtained peptide map The result of FIG. 3 was obtained.

  As shown in FIG. 2, albumin is densely stained in the whole serum (lane 1), but when passing through a liposorber, apoprotein B-100 having a molecular weight of 500,000 and its fragment are removed (lane 2), and an LDL-removed image is obtained. Minutes are obtained (lane 3). Thereafter, when treated with an anion exchange column, the IgG fraction is stained with IgG γ chain and light chain (lane 6), and the obtained IgG-removed fraction is almost free of IgG γ chain and diluted with IgG light chain. (Lane 5). Complement consumption fraction shows the same tendency as LDL-removed fraction (lane 4).

  The results of SDS-PAGE of the sodium urate crystal adsorbed protein from the LDL-removed fraction, complement-consumed fraction, and IgG-removed fraction are lane 9, lane 10, and lane 11, respectively, and there are dark bands around 70 kDa and 25 kDa. It was seen.

  From FIG. 3, all three dark-stained bands of sodium serum urate adsorbed protein in whole serum are “fragments of apolipoprotein B-100”. The 25 kDa band was the immunoglobulin light chain.

  Based on the above results, IgM antibodies can be selectively recovered by using sodium urate crystals after pretreatment for removing LDL from human serum with a liposorber gel.

(Example 2)
A mouse monoclonal antibody was prepared as follows. A hybridoma (Japanese Journal of Urology, Vol. 87, 1120, 1996) producing IgM type (UP509, UP599) and IgG1 type mouse monoclonal antibody (UP720) against human tubular secretory urinary protein, The culture supernatant (RPMI 1640) containing 1% was cultured and the supernatant was collected.

  Liposorber gel was packed in a column, and priming was performed with physiological saline to which a magnesium sulfate injection solution (Japanese Pharmacopoeia) was added so that the Mg concentration became 1 mM. Hydrochloric acid was added to the above hybridoma culture supernatant to adjust the pH to 7, and then passed through a liposorber gel column for pretreatment.

  Sodium urate crystals prepared in the same manner as in Example 1 were washed several times with sodium urate saturated saline, mixed with fiber cellulose CF1 (Whatman), filled into a column, and primed with sodium urate saturated saline. Went.

The culture supernatant passed through the liposorber gel column was passed through a sodium urate crystal column and then washed with 1M NaCl. The IgM type antibody was eluted with 0.5M KH ₂ PO ₄ solution (pH 4.4) and adjusted to pH 7 with NaOH solution to obtain an IgM type antibody.

  The IgM type antibody obtained by the above-mentioned method was analyzed by SDS-PAGE. The results are shown in FIGS.

  From the results of SDS-PAGE of FIG. 4, that is, the mouse monoclonal IgM type antibody (UP509) culture supernatant and the protein eluted from the liposorber gel column and sodium urate crystal column through the culture supernatant, it was adsorbed to sodium urate. The proteins were IgM μ and light chains. As shown in FIG. 5, Ig509 type UP509 and UP599 were bound to sodium urate crystals, but IgG type UP720 was not adsorbed in the mouse antibody.

(Example 3)
Ascitic fluid collected by injecting three types of hybridomas (UP70, UP509, UP599) producing IgM antibodies into the abdominal cavity of BALB / c mice was collected.

  Liposorber gel was packed in a column, and priming was performed with physiological saline to which a magnesium sulfate injection solution (Japanese Pharmacopoeia) was added so that the Mg concentration became 1 mM. The ascites as described above was passed through a liposorber gel column for pretreatment.

  The sodium urate crystals prepared in the same manner as in Example 1 were washed several times with sodium urate saturated saline, mixed with fiber cellulose (CF1: Whatman), filled into a column, and sodium urate saturated saline with Priming was performed.

  Ascites fluid that passed through the liposorber gel column was passed through a sodium urate crystal column and then washed with 1M NaCl.

As a caustic agent, urine was stored in the presence of sodium azide. It was filtered through 1 filter paper, then 5 μm and then 0.22 μm filter. The filtered urine was concentrated using an ultrafiltration membrane with a molecular weight cut off of 30,000. A urine protein concentrate is passed through a sodium urate crystal adsorbed with an IgM type antibody, thoroughly washed with a 1M NaCl solution, and then the IgM type antibody and antigen are eluted with a 0.5M KH ₂ PO ₄ solution (pH 4.4). A mixed sample of IgM type antibody and antigen was obtained.

  The mixed sample of IgM type antibody and antigen obtained by the above method was analyzed by SDS-PAGE. The results are shown in FIG. Furthermore, the result of confirming the adsorption of the antigen by immunoblotting is also shown in FIG.

  The left figure of FIG. 6 shows the results of SDS-PAGE of IgM antibody eluted from urine protein and sodium urate crystal column and CBB staining of antigen. The right figure of FIG. 6 is the immunoblot, UP70 and UP509 are IgM type antibodies recognizing the same antigen with a molecular weight of 30,000 or less, and UP599 is an IgM type antibody recognizing an antigen with a molecular weight of 1 million or more. It was revealed that the IgM type antibody has a high antigen adsorption activity.

  In addition, it was suggested that the sodium urate crystal column through the IgM antibody-containing solution pretreated with a liposorber can adsorb the antigen recognized by the IgM antibody as an affinity column.

Conceptual diagram of operation process It is a figure which shows the result of SDS-PAGE in Example 1. FIG. 2 is a diagram showing the results of LC-MS of sodium urate adsorbed protein in Example 1. It is a figure which shows the result of SDS-PAGE in Example 2. It is a figure which shows the result of SDS-PAGE in Example 2. It is a figure which shows the result of SDS-PAGE in Example 3, and its immunoblot.

Claims (1)

An IgM-type antibody characterized in that an IgM-type antibody is selectively bound to a sodium urate crystal by contacting the IgM-type antibody-containing solution with the sodium urate crystal, and then the IgM-type antibody is dissociated and recovered from the sodium urate crystal. The method for separating and purifying IgM antibodies, comprising a pretreatment in which an IgM antibody-containing solution is brought into contact with an adsorbent formed by immobilizing dextran sulfate.