GB2162856A - Hybridoma capable of producing a monoclonal antibody against bovine lactoferrin - Google Patents

Abstract

A hybridoma capable of producing a monoclonal antibody against bovine lactoferrin is obtained by fusing spleen lymphocytes from a bovine lactoferrin-immunized mouse with mouse myeloma cells. The antibodies are useful for producing high-purity lactoferrin.

Description

SPECIFICATION Hybridoma capable of producing a monoclonal an tibody against bovine lactoferrin This invention relates to a hybridoma capable of producing a monoclonal antibody against bovine lactoferrin which can be advantageously used for the isolation of lactoferrin from milk in a highly pure state and in high yield.

Lactoferrin is an iron-binding protein found in externally secreted fluids such as milk and the like.

It is not only highly beneficial in the feeding of in fants from a nutritional point of view, but also has the physiological effect of exerting a bacteriostatic action on pathogenic bacteria occurring in the in testines and exhibiting a high degree of iron re quirement because of its characteristic iron-binding power. That is, lactoterrin may be said to be an im portant milkprotein which not only serves as a nu trient, but also has a pharmacological significance as an anti-infectious agent, like immunoglobulins and lysozyme present in milk.

Conventionally, in view of the above- described characteristic properties of lactoferrin, a number of methods for separating lactoferrin from milk and purifying it have been proposed. However, since lactoferrin is a protein having a highly reactive mo lecular structure and is also apt to interact with other milkproteins, it has been difficult to isolate a highly pure form of lactoferrin easily and effec tively according to the conventional methods.

For example, one conventional method for sepa rating and purifying lactoferrin comprises provid ing skimmilk from which fat has been separated, removing casein therefrom by isoelectric precipita tion at pH 4.6, salting out the resulting whey frac tion with ammonium sulfate; dialyzing the resulting fraction against deionized water, and then passing the dialysate several times through an ion exchange resin (Gordon et al.: Biochim. Biophys.

Acta, 60, 410-411, 1962; Merton L. Gloves et. al.: Biochim. Biophys. Acta. 100, 154-162, 1965; Jo hansson, B.G. et al.: Acta Chem. Scand., 23, 683, 1969). In addition, modifications of the above-de scribed method include the method in which silica particles are used in place of the ion exchange "resin (Japanese Patent Laid-Open No. 28233/'83) and the method in which, after the dialysate solu tion is passed through an ion exchange resin, the resulting product is further subject to copper affin ity chromatography (Norihiro Kawakata, Yoshio Yoshino, et al., Abstracts of Lectures at the 1983 Annual Meeting of the Japan Biochemical Society, p. 1053). However, these methods are all lacking in practical utility because they require troublesome operations and an unduly long treating time.

Moreover, since lactoferrin has the property of interacting with other proteins present in milk, the above-described methods using an ion exchahge resin cannot avoid contamination of the product with immunoglobulins and other proteins present in milk. Accordingly, it is difficult in practice to iso late a highly pure form of lactoferrin. In addition, these methods are also disadvantageous in that the repeated treatment by salting-out and ion exchange not only causes a marked reduction in the recovery of lactoferrin, but also makes it practically impossible to recover and reuse other milkproteins (than lactoferrin) and other milk components present in the residues obtained during the course of the separation and purification of lactoferrin.

As a result of investigations conducted to solve the above-described problems encountered in the prior art the present inventors have found that a novel monoclonal antibody against bovine lactoferrin can specifically combine with lactoferrin, enabling a highly pure form of the protein to be isolated, and that such a monoclonal antibody against bovine lactoferrin is produced by a hybridoma which can be formed by fusing spleen lymphocytes from a bovine lactoferrin-immunized mouse with mouse myeloma cells.

Accordingly, it is an object of the present invention to provide a hybridoma capable of producing a monoclonal antibody against bovine lactoferrin which can be advantageously used for the isolation of bovine lactoferrin from milk in a highly pure state and in high yield.

Other objects of the present invention will be apparent from the following description.

According to one aspect of the present invention there is provided a hybridoma capable of producing a monoclonal antibody against bovine lactoferrin which can specifically combine with bovine lactoferrin, the hybridoma being obtained by fusing spleen lymphocytes from a bovine lactoferrinimmunized mouse with mouse myeloma cells. In other aspects the invention provides a method of forming the hybridoma, the novel antibodies, and lactoferrin purified therewith.

Thus the hybridoma capable of producing a monoclonal antibody against bovine lactoferrin is obtained by fusing spleen lymphocytes from a bovine lactoferrin-immunized mouse with mouse myeloma cells.

The method of forming hybridomas producing monoclonal antibodies by fusing spleen lymphocytes from an immunized mouse with mouse myeloma cells was first published by G. Kohler and C.

Milstein (Nature 256, 495-497, 1975). Since then, many reports have been made of the formation of hybridomas by cell fusion, as well as scientific researches on the antibodies produced by these hybridomas and their utilization.

However, in applying this general method for the formation of hybridomas to the formation a new hybridoma capable of producing a particular monoclonal antigen, several difficulties are encountered from an operational point of view. Specifically, depending on the type of protein used as an antigen in the formation of a hybridoma, special measures may be required in subsequent procedures for immunization and cell fusion, and influences may be exerted on the screening technique for isolating the desired hybridoma.

The hybridoma capable of producing a monoclonal antibody against bovine lactoferrin according to the present invention can be formed by collecting spleen lymphocytes from the spleen ex cised from a mouse immunized with lactoferrin and fusing these spleen lymphocytes with mouse myeloma cells in the conventional manner. The- method for forming the above-described hybridoma will now be described herein below, Formation of a hybridoma capable of producing a monoclonal antibody against bovine lactoferrjn A human lactoferrin preparation having a purity of 98% is sold by Sigma Co., but bovine lactoferrin is not commercially available. Thus, casein is removed from bovine colostrum by isoelectric precipitation and the resulting whey fraction is treated according to a conventional procedure (for example, Johansson, B.G. et al.: Acta Chem.Scarid., 23, 683, 1969) to obtain a bovine lactoferdin fraction having a purity of about 60%.

Then, a solution of bovine lactoferrin is prepared (usually by using a phosphate buffer salinesolu- tion (PBS), pH 7.2, containing 0.15 M sodium chloride) and mixed with an equal amount of Freund's adjuvant to form an emulsion. By injecting this emulsion into the abdominal cavity, a mouse (usually aged 6 to 8 weeks) is immunized three times at intervals of 2 weeks For this purpose, a highly pure form of bovine lactoferrin must be used.

To this end, it is desirable to raise the purity of bovine lactoferrin by using an antiserum against bovine-immunoglobulins to take up immunoglobulins which are the principal impurities present in the bovine lactoferrin fraction.

If the purity of bovine lactoferrin is low, it is difficult to obtain the desired hybridoma. The reason for This. is that a mouse is more easily immunized with bovine immunoglobulins than with bovine lactoferrin and, therefore, the titer of the antibody against bovine lactoferrin is not fully elevated No particular limitation is placed on the type of mouse used in the present invention. However, it is usually desirable to use a BALB/c strain mouse.

In the prior art, spleen lymphocytes for use in the formation of a hybridoma capable of producing a monoclonal antibody are usually collected from a mouse spleen excised 3 or 4 days after the final immunization with a specific antigen. In the case of bovine lactoferrin to which the present invention is directed, the titer of the antibody observed in the mouse does not reach a maximum 3 or 4 days after the final immunization, and the antibody- producing cells among the spleen lymphocytes are not sufficiently activated at that time. Accordingly, the rate of formation of a desired hybridoma capable of producing a monoclonal antibody against bovine lactoferrin is unsatisfactorily low.

The present inventors have found that the rate of formation of the aforesaid hybridoma can be greatly enhanced by using spleen lymphocytes collected from the spleen excised from a mouse 6 or 7 days after the final immunization with lactoferrin.

The spleen lymphocytes (hereinafter referred to as spleen cells) collected from the mouse spleen in the above-described manner is then fused with mouse myeloma cells (hereinafter referred to as myeloma cells).

No particular limitation is placed on the type of myeloma cells used in the present invention. However,where it is desired to fuse them with spleen cells obtained from a BALBIc strain mouse, it is preferable to use SP2/0-Ag14 cells which do not secrete the K chain of IgG. The fusion may be carried out according to any of thé conventional procedures. However, where SP2/0-Ag14 cells are used as the myeloma cells, it is essential that the fusion time including the addition of a fusion promoter (fusion inducer), mixing and dilution should be in the range of 5 to 15 minutes and preferably 9 to 11 minutes.

If the fusion time is shorter than 5 minutes, the fusion will be incomplete, while if it is longer than 1.5 minutes, the cells will die from poisoning by polyethylene glycol used as a fusion promoter. In this connection, if the fusion time is in the range of 9 to 11 minutes, the rate of colony formation will approach 100%. Where spleen cells from a BALBIc strain mouse immunized with bovine lactoferrin are fused with SP2/0-Ag14 cells, the rate of colony formation (the rate of fusion) can be greatly enhanced by using gas-chromatographic grade polyethylene glycol (M.W. 4000; Merck & Co., Inc.) as a fusion promoter at a concentration of 50%.

After completion of the fusion, the fused cells may be treated in the conventional manner Specifically, the fused cells are dispersed in HT medium (Dulbecco-modified MEM medium containing hypoxanthine, thymidine and 10% fetal calf serum), sprayed over a 96-hole micro titer plate and cultured at a temperature of 37"C under an atmosphere of 5% carbon dioxide. From the following day, selection of hybridomas is carried out in HAT medium (Dulbecco- modified MEM medium containing hypoxanthine, amino- pterine, thyr?idine and 10% fetal calf serum).

As soon as the colonies have grown to a sufficient size, the hybridomas are screened according to the solid phase method. Then, the hybridomas exhibiting a positive reaction are cloned according to the limiting dilution method.

In one embodiment, the solid phase method is carried out by causing a soluble antigen to be adsorbed to a 96-hole soft microwell, treating the wells with bovine serum albumin (BSA) to block the portions to which the antigen is not adsorbed, and placing the supernatants of the aforesaid culture media in the wells to effect reaction with the antigen. Upon completion of the reaction, the wells are washed thoroughly and antibodies against biotinylated mouse antibodies are added thereto assecondary antibodies. Thereafter, the wells are treated with avidin and fluorescein-labeled biotin to detect the desired antibody by the production of fluorescence.

The soluble antigen used for this purpose must be bovine lactoferrin having as high a purity as possible. The reason for this is that, if the purity of bovine lactoferrin is low, the hybridomas for impurity antigens also exhibit a positive reaction and this makes it difficult to find out a desired hybridoma producing a monoclonal antibody against bovine lactoferrin.

In one embodiment, the limiting dilution method is carried out as follows: A dispersion of 108 mouse thymus cells and 50 hybridoma cells in 10 ml of HT medium is sprayed over a 96-hole micro titer plate so that one or less hybridoma cell will be present in each well and, hence, single colonies of the hybridoma will be formed. The mouse thy mus cells are added as feeder cells because hybridoma cells cannot grow at low cell densities.

The above-described cloning procedure is re peated three or more times to obtain a mono cloned hybridoma.

Then, the resulting hybridoma capable of pro ducing a monoclonal antibody against bovine lactoferrin may be grown in the usual manner.

Specifically, the hybridoma is injected into the ab dominal cavity of mice and their ascitic fluid is re covered. Alternatively, the hybridoma is cultured in a suitable medium and its supernatant is re covered. The recovered ascitic fluid or supernatant is then purified by precipitation with ammonium sulfate and ion exchange chromatography to obtain the monoclonal antibody against bovine lactoferrin.

In one embodiment, the monoclonal antibody against bovine lactoferrin can be prepared as fol lows: Cells of the aforesaid hybridoma are dis persed in a phosphate buffer saline solution (PBS) and injected into the abdominal cavity of BALBIc strain mice to which pristan (2,6,10,14-tetramethyl pentadecane) has previously administered. After 7 to 10 days, their ascitic fluid is collected, cleared by centrifugation, diluted with PBS so as to give a protein concentration of the order of 10 to 12 mg/ ml, and then salted out with 45% saturation am monium sulfate. A solution of the resulting precipitate fraction is dialyzed and then purified by ion exchange chromatography.

Then, the monoclonal antibody against bovine lactoferrin thus obtained may be used in the sepa ration and purification of bovine lactoferrin. This can be accomplished by preparing an affinity chromatographic column of the antibody according to the procedure described below and passing a solution of crude bovine lactoferrin separated from milk through the affinity-chromatographic column.

Preparation of an affinity-chromatographic column The monoclonal antibody against bovine lacto iferrin prepared in the above-described manner is mixed with an equal amount of an insoluble carrier for use in affinity chromatography [for example, Affigel-lO; Bio-Rad Co.] and this mixture is stirred at low temperature to bind and fix the antibody to the carrier. After washing, those functional groups on the carrier to which the aforesaid antibody is not attached are inactivated and the carrier is then washed. The resulting affinity gel is packed into a column of suitable size to form an affinity- chro matographic column. Crude bovine lactoferrin obtained from milk is dissolved in a phosphate buffer saline solution (PBS) and the resulting solution is passed through the aforesaid column.Then, the affinity gel within the column is washed with PBS to remove the unadsorbed fraction, followed by thor ough washing with PBS containing 0.5M sodium chloride and then 0.15M sodium chloride solution.

Thereafter, the adsorbed bovine lactoferrin is eluted with an acetate buffer solution, pH 2.7, containing 0.15M sodium chloride. The eluate fraction containing bovine lactoferrin is adjusted to pH 7, dialyzed against deionized water and then freezedried to obtain bovine lactoferrin having a purity of 98% or greater.

The present invention is further illustrated by the following example.

Example Using an ion exchanger resin, a bovine lactoferrin fraction was separated from bovine colostrum according to the conventional method (Johansson, B.G. et al.: Acta Chem. Scand., 23, 683, 1969). This bovine lactoferrin fraction had a purity of 60%.

To the above bovine lactoferrin fraction was added an antiserum against bovine immunoglobulins. Thus, bovine immunoglobulins constituting the principal impurities were taken up to raise the purity of bovine lactoferrin to about 90%.

The bovine lactoferrin thus obtained was dissolved in a phosphate buffer saline solution (PBS), pH 7.2, containing 0.15 M of sodium chloride so as to give a concentration of 0.3%. This solution was mixed with an equal amount of Freund's complete adjuvant (Difco) to form an emulsion. A BALBIc strain mouse, aged 6 to 8 weeks, was immunized by injecting the emulsion into the abdominal cavity thereof. This immunization was repeated three times at intervals of 2 weeks. Seven days after the final immunization, the spleen was excised from the mouse and spleen cells were collected therefrom. These spleen cells were dispersed in DMEM medium (Dulbecco- modified minimum essential medium) and mixed with SP2/0-Ag14 mouse myeloma cells in a ratio of 2:1.To this mixture was added a 50% solution of polyethylene glycol (gaschromatographic grade, M.W. 4000; Merck & Co., Inc.) as a fusion promoter. Thus, fusion of the cells was completed in 10 minutes. The resulting fused cells were separated from polyethylene glycol by centrifugation, dispersed in HT medium so as to give a cell density of 1 x 107 cells/ml or less, sprayed over 96-hole micro titer plates, and cultured at 37 C under an atmosphere of 5% carbon dioxide. From the day after the commencement of the culture (i.e., the first day), half the medium was continually exchanged with HAT medium. On the 17th day, hybridomas were screened according to the solid phase method. As a result, 100% of the hybridomas formed a colony and 8.3% of the hybridomas exhibited a positive reaction for bovine lactoferrin.

Then, the hybridomas exhibiting a positive reaction were transferred to 24-hole micro titer plates and cloned as soon as they grew to a cell density of 1 x 105 cells/ml. Thereafter, they were cultured in HT medium for 2 weeks and the hybridomas forming a single colony in the well were subjected to secondary cloning.

This cloning and screening procedure was repeated three times. Thus, there were obtained monoclones of a hybridoma capable of producing an antibody against bovine lactoferrin.

A stock of the resulting hybridoma capable of producing a monoclonal antibody against bovine lactoferrin is maintained in the American Type Cul tureCollection, 12301 Parklawn - Drive, Rockvillet Maryland 20852-1776 with Deposition No. ATCC HB 8852.

Claims (11)

1. A hybridoma capable of producing a monoclonal antibody against bovine lactoferrin which can specifically combine with bovine lactoferrin, the hybridoma being obtained by fusing spleen lymphocytes from a bovine lactoferrin-immunized mouse with mouse myeloma cells.

2. A hybridoma as claimed in claim 1 whereinthe spleen lymphocytes are collected from a mouse spleen excised 6 or 7 days after the final immunization.

3. A hybridoma as claimed in claim 1 or claim 2 wherein the mouse myeloma cells are SP2/0-Ag14 cells. -

4. A method for forming a hybridoma capable of producing a monoclonal antibody against bovine lactoferrin which comprises the steps of providing a solution of bovine lactoferrin separated from the whey fraction of milk; immunizing. a mouse with an emulsion formed by mixing equal amounts of the lactoferrin solution and Freund's complete adjuvant; collecting spleen lymphocytes from the spleen of the mouse; fusing the spleen lymphocytes with mouse myeloma cells and culturing the resulting fused cells; selecting the formed hybridomas in HAT medium; screening the selected hybridomas according to the solid phase method to obtain a hybridoma exhibiting a positive reaction; and subjecting the hybridoma to a cloning procedure for obtaining a monoclone of the hybridoma.

5. A method as claimed in claim 4 wherein said solution of bovine lactoferrin is prepared by separating bovine lactoferrin from the whey fraction of bovine colostrum, adding an antiserum against bovine immunoglobulins to the bovine jactoferrin to take up and remove the bovine immunoglobulins and other impurities present therein, and dissolving the resulting purified bovine lactoferrin in a phosphate buffer saline solution (PBS).

6. A method as claimed in claim 4 or claim 5 wherein spleen lymphocytes collected from a mouse spleen excised 6 or 7 days after the final immunization with bovine lactoferrin are used as said spleen lymphocytes, SP2/0-Ag14 cells are used as said mouse myeloma cells, and these cells are fused over a period of 5 to 15 minutes and preferably 9 to 11 minutes.

7. Hybridoma substantially as herein described and exemplified.

8. Method of producing hybridoma substantially as herein -described and exemplified.

9. Monoclonal antibody to bovine lactoferrin.

10. Monoclonal antibody to bovine lactoferrin, produced by the hybridoma of claims 1, 2, 3 or 7.

11. Bovine lactoferrin isolated by use of the monoclonal antibody of claim 9 or claim 10.