JPH0557997B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for separating and purifying human lactoferrin from human milk.

Lactoferrin is an iron-binding protein present in exocrine fluids such as milk, and is not only extremely beneficial nutritionally during infant lactation, but due to its iron-binding properties, it is also a protein that is associated with iron-requiring pathogens in the intestinal tract. It has the physiological effect of exhibiting a strong bacteriostatic effect against sexually transmitted bacteria. In other words, lactoferrin, together with immunoglobulin and lysozyme present in milk, is a milk protein that is important not only nutritionally but also pharmacologically as an infection-preventing substance.

Prior Art In view of the above-mentioned characteristics of lactoferrin, various methods have been proposed for separating and purifying lactoferrin from milk. However, since lactoferrin is a protein with a highly reactive molecular structure and also interacts with other milk proteins, conventional methods cannot easily and effectively separate and purify lactoferrin to collect it. It was difficult to do so.

For example, conventionally, skimmed milk, in which fat is separated from milk,
Casein was removed by isoelectric precipitation at pH 4.6, and the whey fraction obtained was then salted out with ammonium sulfate. The fraction was dialyzed against pure water and passed through an ion exchange resin several times for separation. Method of purification (Gordon, Ziegler, Bash et al.: “Biochim.
Biophys. Acta, 60, 410-411, 1962” and
Merton L. Grove et al.: “Biochim. Biophys.
Acta 100, 154-162, 1965” or Johansson,
BGet al.: “Acta Chem.Scand.23, 683,
1969'') and a method using silica particles instead of ion exchange resin in the above method (Japanese Patent Laid-Open No. 58-28233).
No.) and a method of further performing copper affinity chromatography treatment after passing through an ion exchange resin as described above (Norihiro Kawakata, Yoshio Yoshino, et al., Proceedings of the Biochemical Society of Japan, p. 1053, 1983). However, these methods are difficult to operate and require a long processing time, so they are not practical.

Furthermore, because lactoferrin has the property of interacting with other proteins present in milk, the above method using an ion exchange resin cannot avoid contamination with other proteins such as immunogallobulin in milk. Therefore, it is practically difficult to collect lactoferrin with high purity, and in addition, because the salting out and ion exchange treatments are repeated, a significant decrease in the recovery rate of lactoferrin is unavoidable. However, there is a drawback that it is virtually impossible to recover and reuse milk proteins other than lactoferrin and other milk components contained in the residue obtained in the process of separating and purifying lactoferrin.

Purpose of the Invention The present invention has been made in view of the current state of the prior art regarding lactoferrin, and is intended to advantageously separate and purify highly physiologically active human lactoferrin from human milk with high purity and high yield. The purpose is to provide a method for

The present invention will be explained in detail below.

Structure of the Invention The structural feature of the present invention is that human lactoferrin is separated and purified from human milk using an affinity column comprising a monoclonal anti-human lactoferrin antibody immobilized on an insoluble carrier. .

That is, the method of the present invention is a method for separating and purifying lactoferrin that utilizes biological affinity, which is an extremely specific interaction between biological components, and is a method for separating and purifying lactoferrin using biological affinity, which is a method for separating and purifying lactoferrin that utilizes conventional separation methods that utilize physical and chemical affinity. It is essentially different.

The monoclonal anti-human lactoferrin antibody used in the present invention is a monoclonal anti-human lactoferrin antibody obtained by fusing mouse spleen lymphocytes (splenocytes) immunized with human lactoferrin with mouse myeloma cells (myeloma cells). A ferrin antibody-producing hybridoma is administered intraperitoneally to a mouse according to a known method, and the ascites is collected, or the hybridoma is cultured in a medium and its supernatant is collected, and the supernatant is collected. Obtained by purifying the liquid using ammonium sulfate precipitation and anion exchange resin.

Next, a method for forming the above hybridoma that produces the monoclonal anti-human lactoferrin antibody used in the present invention will be explained.

Formation of monoclonal anti-human lactoferrin antibody-producing hybridoma A solution of human lactoferrin is prepared (generally using phosphate buffered saline (PBS) PH7.2 containing saline),
Mice (generally 6 to 8 weeks old) are intraperitoneally immunized three times at two-week intervals with an emulsion obtained by mixing equal amounts of this solution and Freund's adjuvant. Although the human lactoferrin used in this case may contain some impurities, it is of course preferable to use one with high purity in order to obtain the desired hybridoma.

The type of mouse used in the present invention is not particularly limited, but BALB/c mice are generally preferred.

Conventionally, splenic lymphocytes used to form monoclonal antibody-producing hybridomas are generally collected from the spleen removed 3 to 4 days after the final immunization with a specific antigen. In the case of lactoferrin, the increase in antibody titer in mice did not reach its maximum value 3 to 4 days after the final immunization, and the activation of antibody-producing cells in the spleen was insufficient. , the formation rate of the desired monoclonal anti-human lactoferrin antibody-producing hybridoma becomes low.

The present inventors have found that the formation rate of the above-mentioned hybridomas is extremely high when splenic lymphocytes collected from spleens removed from mice 6 to 7 days after the final immunization with human lactoferrin are used.

Splenic lymphocytes (hereinafter referred to as splenocytes) collected from the mouse spleen as described above are fused with mouse myeloma cells (hereinafter referred to as myeloma cells).

The myeloma cells used here are not particularly limited, but when fused with BALB/c mouse splenocytes, SP2/0-, which does not secrete the κ chain of IgG, may be used.
Preferably, Agl4 is used. The fusion method can be carried out according to known methods, but when using the above SP2/0-Ag14 as myeloma cells, the fusion time, which consists of adding, mixing, and diluting a fusion promoter (fusion inducer), is 5-15 minutes, preferably 9
It is important to stay within the range of ~11 minutes.

In this case, if the fusion time is shorter than 5 minutes, the fusion will be incomplete, while if it exceeds 15 minutes, the cells will die due to poisoning by the polyethylene glycol used as the fusion promoter. Incidentally, when the above-mentioned fusion time is set in the range of 9 to 11 minutes, a colony formation rate close to 100% can be obtained. We also immunized with human lactoferrin.
BALB/c mouse splenocytes and SP2/0-Ag14
In the case of fusing, a particularly high colony formation rate (fusion rate) can be obtained by using polyethylene glycol with a molecular weight of 4000, manufactured by Merck & Co., Ltd. for gas chromatography, at a concentration of 50% as a fusion promoter.

After the fusion is completed, the fused cells are processed using the conventional method.
Dispersed in HT medium (Dulbetzko's modified MEN medium containing hypoxanthine, thymidine, and 10% fetal bovine serum), then spread on a 96-well microtiter plate, and cultured at 37°C in a 5% carbon dioxide atmosphere. Then, from the next day, hybridomas are selected in HAT medium (Dulbetzko's modified MEM medium containing hypoxanthine, aminopterin, thymidine, and 10% fetal bovine serum).

Next, when the hybridoma colony has grown sufficiently large, the solid phase method is used.
Hybridomas that show a positive reaction are screened using the limiting dilution method and cloned using the limiting dilution method.

Note that the solid phase method uses soluble antigens.
After adsorption to a 96-well soft microwell, the non-adsorbed areas in the wells were blocked with bovine serum albumin (BSA), and the culture supernatant was placed in each well to react with the antigen. The wells are thoroughly washed, then an antibody against a bithionylated mouse antibody is added as a secondary antibody, and then adipine and biotin labeled with a fluorescent dye are reacted to detect the target antibody using fluorescence. Ta.

In addition, in the limiting dilution method, ¹⁰⁸ mouse thymocytes and 50 hybridomas are dispersed in 10ml of HT medium, and then dispersed onto a 96-well microtiter plate so that there is no more than one hybridoma per well. This was done to obtain a single colony of hybridomas. Note that mouse thymocytes were added as feeder cells since hybridomas cannot proliferate at low cell density.

Cloning as described above is repeated three or more times to obtain monocloned hybridomas.

In the present invention, the monoclonal anti-human lactoferrin antibody produced by the hybridoma formed as described above is transferred to an insoluble carrier such as a carrier for affinity chromatography, for example, Afuigel-10 (Bio-Rad) and known means. It is then bound and fixed, and used as an affinity column for the separation and purification of human lactoferrin from human milk.

However, monoclonal anti-human lactoferrin antibodies immobilized on a carrier such as affinity gel can be dispersed in phosphate buffer (PBS) pH 7.2 containing 0.02% sodium azide. ℃
It can be stored for about 3 months or more at a temperature of .

In order to separate and purify human lactoferrin from human milk using an affinity column in which a monoclonal anti-human lactoferrin antibody is immobilized on an insoluble carrier as described above, the affinity column is heated at 50°C or lower. After passing an appropriate amount of a human lactoferrin-containing sample such as human milk at a temperature of, preferably room temperature, and flowing out the non-adsorbed fraction to the column,
The column is washed with a phosphate buffer containing 0.5M NaCl at pH 7.2 and a saline solution containing 0.15M, and then passed through an acetate buffer containing 0.15M NaCl at a pH below 4.7, preferably between PH 4.3 and 2.7. It is recommended to elute human lactoferrin adsorbed on the column by Immediately add an alkaline agent to the obtained human lactoferrin fraction to return the pH to neutral.

According to the present invention, human lactoferrin is separated and purified from human milk using an affinity column immobilized with a monoclonal anti-human lactoferrin antibody. , there is no need for pretreatment such as dialysis or freeze-drying,
In addition, in the conventional method, it is necessary to pass the above-mentioned processed product through an ion exchange resin several times, but with the present invention, human lactoferrin can be separated from human milk by passing it through an affinity column only once. It can be easily separated from the components. In addition, the purity of lactoferrin obtained by conventional methods is about 66% at maximum,
The recovery rate is also about 60%, but according to the present invention, extremely high purity human lactoferrin with a purity of 98% or more can be obtained by passing liquid through the column once, and the recovery rate is high at 80% or more to 90% or more. It is possible to collect it.

Unfortunately, the purity and recovery rate of human lactoferrin were calculated by the following procedure.

When the collected human lactoferrin fraction is subjected to SDS electrophoresis, a complete single band is obtained, but human milk contains a selector component (SC) with approximately the same molecular weight (approximately 80,000) as lactoferrin. 100% from the results obtained only by electrophoresis because it contains a glycoprotein called
Lactoferrin cannot be determined. Therefore, anti-SC
Since no sedimentation line was observed when double immunodiffusion of serum and human lactoferrin fraction was performed, the SC content that may be contained in the human lactoferrin fraction is at most 0.01% or less. You can say that.
That is, for the above sedimentation line to occur, the human lactoferrin fraction must contain at least 0.01% SC.

Therefore, based on the above-mentioned SDS electrophoresis, immunodouble diffusion, and measurement of the protein concentration of the human lactoferrin fraction, the purity of human lactoferrin obtained by the present invention was determined to be 98% or more.

Regarding the recovery rate, it was confirmed that no sedimentation line was generated when double immunodiffusion was performed between anti-human lactoferrin serum and the non-adsorbed fraction. It can be determined that the residual content of human lactoferrin is at most 0.01% or less.
That is, for the above sedimentation line to occur, the non-adsorbed fraction must contain at least 0.01% human lactoferrin.

Therefore, it can be determined that the recovery rate of human lactoferrin according to the present invention is at least 80% or more or 90% or more based on the above-mentioned immune double diffusion and protein concentration measurements.

As mentioned above, the present invention utilizes the biological affinity for an adsorbent (affinity column) on which a specific antibody is immobilized to advantageously combine human lactoferrin as a target substance with other milk components. It is something that separates. In other words, the present invention utilizes the reactivity of an antigen (human lactoferrin) and an antibody (monoclonal anti-human lactoferrin antibody), which have biologically specific affinity for each other. - Since it utilizes the extremely high specificity of antibody reaction, highly pure human lactoferrin can be separated in a single step.

Unfortunately, the method of separating a target substance using an affinity column that utilizes an antigen-antibody reaction is widely known. In the method of using antiserum without using monoclonal antibodies, the antiserum against the target substance is obtained and the antibody contained therein is bound to a carrier, and the target substance can be recovered with high purity. I can't. This is because antiserum contains many types of antibodies in addition to antibodies against the target substance, and this is especially true if the antigen used for immunization to obtain the antiserum contains impurities. This is because antibodies against impurities are also present.

In other words, to recover the target substance with high purity,
It is necessary to use a column in which a monoclonal antibody that recognizes only the target substance is bound to a carrier, and in order to recover a large amount of the target substance, a large amount of antibody is naturally required. While antiserum is limited in quantity, monoclonal antibodies can be obtained in unlimited quantities as long as the hybridomas that produce them are formed.

In addition, in the conventional method of separating and purifying a target substance using an affinity column, the target substance can be obtained with high purity and yield, but when releasing the target substance bound to an antibody, it is generally difficult to PH
However, according to the present invention, the elution pH is This method has the advantage of being able to overcome these drawbacks because it can be eluted even at a slightly higher pH than conventional methods, and the PH of the elution fraction containing the target substance is returned to neutral immediately after elution. .

Furthermore, the lactoferrin separated and purified by the method of the present invention also has the following advantages.

As mentioned above, lactoferrin has a strong physiological effect of inhibiting the growth of iron-requiring pathogens due to its iron-binding ability, and therefore it must be recovered to utilize its physiological effect. The lactoferrin obtained must not be iron-saturated and must have iron-binding ability. However, since the lactoferrin recovered by conventional methods is an iron-bound lactoferrin that sometimes exhibits a pink color (see JP-A-58-28233),
Unless iron is removed from lactoferrin using an iron chelating substance such as EDTA, the above-mentioned physiological effects cannot be expected.

In contrast, lactoferrin separated and purified according to the method of the present invention completely retains its original iron-binding ability. Incidentally, the iron-binding ability of human lactoferrin obtained by the method of the present invention and commercially available human lactoferrin (manufactured by Sigma, purity 98%) was determined by the method of Suzuki and Nonaka et al. As a result of measurement according to the Japanese Journal of Human Lactoferrin, Vol. 31, No. 4, 395-403, 1978), both showed an iron binding capacity of 16 mg per 1 g of human lactoferrin. It is demonstrated that ferrin retains its full native iron binding capacity.

Therefore, human lactoferrin obtained by the present invention can be applied as a prophylactic agent for preventing infections in infants caused by iron-requiring pathogens, and as a therapeutic agent for various symptoms caused by the above-mentioned pathogens. In addition, it can be added to powdered milk for infants to make the protein composition similar to that of breast milk, and it also has the effect of increasing the above-mentioned infection prevention ability of the powdered milk.

EXAMPLES The present invention will be explained in more detail with reference to Examples below.

Example 1 Formation of monoclonal anti-human lactoferrin antibody-producing hybridoma Human lactoferrin (manufactured by Sigma, purity 98%)
phosphate buffered saline (PBS) containing 0.15M NaCl PH
7.2 to a concentration of 0.3%, and an equal amount of Freund's adjuvant (manufactured by Difco) was mixed with this solution to make an emulsion. It was administered intravenously and immunized. This immunization was carried out three times at two-week intervals, and 7 days after the final immunization, the spleen was removed from the mouse and splenocytes were collected from it. These splenocytes were cultured in DMEM medium (Dulbetzko's modified MEM medium).
and mixed with mouse myeloma cells SP2/0-Ag14 at a ratio of 2:1. A 50% polyethylene glycol (manufactured by Merck & Co., Ltd., for gas chromatography, molecular weight 4000) solution was added as a fusion promoter to this mixed solution, and fusion was completed in 10 minutes. The thus obtained fused cells were centrifuged to remove polyethylene glycol, and then placed in HT medium 1x.
Distribute to a cell density of ¹⁰⁷ cells/ml or less,
Then, plate in a 96-well microtiter plate and
Cultivation was started at a temperature of .degree. C. in a 5% carbon dioxide atmosphere. From the day after the start of culture (first day), half of the medium was replaced with HAT medium, and on the 17th day, screening was performed using the solid phase method. The hybridoma colony formation rate was 99%, and the human lactoferrin positive rate was 24%.

Next, the hybridomas that showed a positive reaction were transferred to a 24-well microtiter plate and cultured in HT medium, and cloning was performed when the cell density increased to 1×10 ⁵ cells/ml. Thereafter, the cells were cultured in HT medium for 2 weeks, and wells forming a single colony were selected for secondary screening.

Such cloning and screening operations were performed three times in total to obtain a monoclone of a hybridoma that produced an anti-human lactoferrin antibody.

Preparation of monoclonal anti-human lactoferrin antibody The monoclonal anti-human lactoferrin antibody-producing hybridomas formed as described above were dispersed in PBS at a rate of 10 x ⁷ cells/0.5 ml per mouse, and pre-injected with pristane (Pristan; 2,6,
10,14-tetramethylpentadecane) was injected intraperitoneally into 10 BALB/c mice.

7 to 10 days after the above injection, 46 ml of ascites was collected.
Cleaned by centrifugation to a protein concentration of 10-12
Dilute with PBS to give mg/ml, then 45%
Salting out was carried out with saturated ammonium sulfate. The obtained precipitate fraction was 0.04M
After dissolving in 0.02M Tris buffer (PH7.9) containing sodium chloride, it was dialyzed against the buffer overnight at low temperature. Pack 200 ml of the obtained dialysis fluid into a column with a diameter of 2 cm and a height of 80 cm, containing 0.04 M to 0.15 M of salt.
Ion exchange chromatography was performed using an ionic strength gradient using 0.02M Tris buffer (PH7.9), and fractions eluted at 0.06 to 0.1M salt concentration were collected. This fraction was further precipitated with 50% saturated ammonium sulfate, and the resulting precipitate was dissolved in deionized water, dialyzed, and lyophilized to obtain 260 mg of monoclonal anti-human lactoferrin antibody.

In addition, ELISA method (Enzyme linked
The type of antibody was confirmed by immunosorbent assay and was found to be IgG.

Preparation of affinity column Purified monoclonal anti-human lactoferrin antibody obtained as described above 10-30 mg/ml, preferably
More than 20 mg/ml was combined with an equal amount of affinity chromatography support Affigel-10 at low temperature overnight with stirring, and washed several times with 0.1 M sodium bicarbonate buffer at pH 8.0 containing 0.15 M NaCl. After that, with hydrochloric acid
The mixture was mixed with an equal amount of 0.1M ethanolamine adjusted to pH 8.0, and gently stirred at room temperature for 1 hour to inactivate the functional groups on the carrier that could not adsorb the monoclonal anti-human lactoferrin antibody. after that,
Phosphate buffer solution (PBS) with PH7.2 containing 0.15M NaCl
The carrier was thoroughly washed with water and packed into a column of an appropriate size to prepare an affinity column.

Separation and Purification of Human Lactoferrin A column with an inner diameter of 13 mm was filled with 3 ml of the affinity gel on which the monoclonal anti-human lactoferrin antibody prepared as described above had been immobilized. In addition, 6.6 mg of monoclonal anti-human lactoferrin antibody was immobilized per ml of this affinity gel. Next, after thoroughly washing the affinity gel in the column with PBS, 7 mg of human lactoferrin (manufactured by Sigma, purity 98%) was added to 2 ml of PBS.
A solution dissolved in the above was flowed through the column at a rate of 1.2 ml/min.

Next, wash the affinity gel in the column with PBS.
The non-adsorbed fraction was removed. At this time, almost no protein elution was observed, and only a peak corresponding to 2% of impurities in the sample solution was obtained. Furthermore, after thoroughly washing the affinity gel with a phosphate buffer of PH 7.2 containing 0.5M salt and a 0.15M salt solution, the adsorbed human lactoferrin was removed with an acetate buffer of PH 2.7 containing 0.15M salt. It was eluted by Note that these series of operations were performed at room temperature. After adjusting the pH of this elution fraction to 7, it was dialyzed against deionized water and lyophilized to obtain 6.5 mg of human lactoferrin. The purity of the obtained human lactoferrin is over 98%,
16 mg of iron was bound per gram. That is, as seen in this example, the affinity column used in the present invention can recover human lactoferrin in high yield without impairing its purity or iron-binding properties.

Example 2 The affinity gel in the used column (i.e., repeated use of the column) in Example 1 was reduced to 0.5M.
After thorough washing with phosphate buffer of pH 7.2 containing salt and PBS, 2.5 ml of defatted human colostrum was added to the column.
was run in a similar manner to that described in Example 1.

However, the elution is at pH 4.3 containing 0.15M salt.
It was carried out in 0.001M acetate buffer. As a result, a fraction adsorbed to affinity gel and a non-adsorbed fraction were obtained. Human lactoferrin was recovered from the adsorbed fraction in the same manner as described in Example 1 to obtain 7.5 mg. The purity of the recovered human lactoferrin is 98
%, the iron binding capacity was 15 mg/g.

As seen in this example, according to the present invention, highly purified human lactoferrin can be recovered from human milk.

Claims (1)

[Claims] 1. Human milk is treated with an affinity column comprising a monoclonal anti-human lactoferrin antibody immobilized on an insoluble carrier, and human lactoferrin in the human milk is adsorbed onto the column. , this adsorbed human lactoferrin is kept at a pH of 4.7 or less, preferably from 4.3 to 2.7.
A method for separating and purifying human lactoferrin from human milk, the method comprising eluating human lactoferrin using a buffer solution and immediately returning the pH of the eluted lactoferrin fraction to neutral. 2. The monoclonal anti-human lactoferrin antibody is produced by a hybridoma obtained by fusing mouse spleen lymphocytes immunized with human lactoferrin with mouse myeloma cells. Claim 1 Method described. 3. The method according to claim 1, wherein the column is washed with a neutral pH buffer before elution treatment of human lactoferrin. 4 Wash the column sequentially with a neutral PH phosphate buffer containing 0.5M saline and a 0.15M saline solution,
Elution of human lactoferrin at pH containing 0.15M NaCl
Claim 3 carried out using the acetate buffer of 4.3 to 2.7
The method described in section.