FR2567539A1 - HYBRIDOME CAPABLE OF PRODUCING MONOCLONAL ANTIBODY AGAINST BOVINE LACTOFERRIN AND PROCESS FOR PREPARING THE SAME - Google Patents

Abstract

THE OBJECT OF THE INVENTION IS A HYBRIDOMA CAPABLE OF PRODUCING A MONOCLONAL ANTIBODY AGAINST BOVINE LACTOFERRIN, WHICH HYBRIDOMA IS OBTAINED BY FUSING RATE LYMPHOCYTES FROM A MOUSE IMMUNIZED BY BOVINE LACTOFERRIN, SELLOCYEL CELLS RATE BEING PREFERREDLY COLLECTED FROM A MOUSE RATE EXCISE 6 OR 7 DAYS AFTER FINAL IMMUNIZATION AND MOUSE MYELOMA CELLS PREFERREDLY FROM SP2O-AG CELLS 14. APPLICATION TO OBTAINING MONOCLONAL ANTIBODIES AGAINST BOVINE LACTOFERRIN, USED IN THE SEPARATION AND PURIFICATION OF BOVINE LACTOFERRIN.

Description

I Hybridoma capable of producing a monoclonal antibody

  bovine lactoferrin and proceeded to prepare it for

  The invention relates to a hybridoma capable of producing a monoclonal antibody against lactoferrin

  bovine, which can advantageously be used for

  solemnly lactoferrin from milk in a

  very pure state and with a high yield.

  Lactoferrin is a protein that binds iron

  found in fluids secreted by ex-

  dull, such as milk and the like. She is not

  only highly beneficial for the food of

  from a nutritional point of view but also

  the physiological effect of exerting a bacterial

  riostatic on the pathogenic bacteria that are found in the intestines and require a high degree of iron because of its characteristic ability to bind iron. More specifically, we can say that lactoferrin

  is an important milk protein that does not serve only

  nutrient but also presents

  pharmacological significance as an anti-

  as immunoglobulins and lyso-

zyme present in the milk.

  In a classical way, because of the properties

  As described above for lactoferrin, a number of methods have been proposed for separating lactoferrin from milk and purifying it. However, since lactoferrin is a protein having

  a highly reactive molecular structure and is also

  able to interact with other milk proteins,

  it was difficult to isolate easily and effectively

  a highly pure form of lactoferrin according to the

classical processes.

  For example, a conventional method for separating and purifying lactoferrin includes the use of skimmed milk from which the fat has been separated, the removal of casein therefrom by isoelectic precipitation at pH 4.6, the release of the resulting whey fraction using ammonium sulphate, the

  dialysis of the resulting fraction against water

  ionized, then passage of 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); Johansson, B.G. et al .: Acta Chem. Scand. 23

  683, (1969)]. In addition, modifications of the process de-

  Criteria above include the method according to which silica particles are used instead of the ion exchange resin (JA 28233/83) and the method according to which, after the dialysate solution has passed through an ion exchange resin, the resulting product is further subjected to affinity chromatography with copper (Norihiro Kawakata, Yoshio Yoshino et al., Abstracts

  of Lecture at the 1983 Annual Meeting of the Japan Bio-

  Chemical Society, p. 1053). However, these methods are

  all of them of no practical use because they require

  complicated operations and the duration of

excessively long.

  In addition, since lactoferrin

  the property of interacting with other proteins

  in the milk, the processes described above use

  ion exchange resin can not prevent contamination of the product with immunoglobulins and

  other proteins present in the milk. As a result

  it is difficult in practice to isolate a high form

  pure lactoferrin. In addition, these methods are also disadvantageous in that the treatment

  repeated by release and ion exchange not only causes

  a marked reduction in lake recovery.

  toferrin, but also makes it virtually impossible to recover and reuse other proteins from

  milk (other than lactoferrin) and other

  of milk present in the residues obtained during

  in the course of separation and purification

lactoferrin.

  Investigations to solve the above-described problems encountered in prior art methods for isolating a highly pure form

  of lactoferrin from milk led to the

  to find that a monoclonal antibody against bovine lactoferrin can be specifically combined with lactoferrin and that such a monoclonal antibody

  against bovine lactoferrin is produced by a hybri-

  dome that can be formed by fusing spleen lymphocytes of a mouse immunized against lactoferrin

  bovine, with mouse myeloma cells.

  Accordingly, one of the objects of this

  The invention is to provide a hybridoma capable of producing

  a monoclonal antibody against bovine lactoferrin

  ne, which can be advantageously used for the isolation

  bovine lactoferrin from milk in

  a state. highly pure and with a high yield.

  Other objects of the present invention

  will read the description which follows.

  These objects and other objects still in the pre-

  The present invention is obtained by a hybridoma capable of producing a monoclonal antibody against bovine lactoferrin, which can be specifically combined with

  bovine lactoferrin, the hybridoma being obtained in

  spleen lymphocytes from a mouse

  immunized against bovine lactoferrin, with

  mouse myeloma, as well as a method for

sea hybridoma.

  The subject of the invention is a hybridoma capable of producing a monoclonal antibody against bovine lactoferrin, which is obtained by fusion of lymphocytes of

  spleen from a mouse immunized against lactobacillus

  bovine ferrin with mouse myeloma cells,

  and a method for forming the hybridoma.

  The process for the formation of hybridomas

  monoclonal antibodies by fusion of spleen lymphocytes from a mouse immunized with

  mouse myeloma cells was published for the first time

  G. Kahler and C. Milptein (Nature, 256, 495-497 (1975)) 7. Since then, many reports have been made of hybridoma formation by

  cells and related scientific research

  the antibodies produced by these hybridomas and their use.

  However, when applying this general process

  neral for the formation of hybridomas for the formation of a

  new hybridoma capable of producing a monoclonal antibody

  clonal, there are various difficulties

  from an operational point of view. Specifically, depending on the type of protein used as antigen

  in the formation of a hybridoma, special measures

  may be necessary in the sub-

  sequences for immunization and cell fusion, and influences can be exerted on the screening technique for the isolation of the hybridoma

longed for.

  The hybridoma capable of producing a

  noclonal anti-bovine lactoferrin according to the present invention can be formed by collecting lymphocytes

  of spleen from the excised spleen from a

  immunized with lactoferrin and merging these

  spleen lymphocytes with myeloma cells from

  laugh in the classic way. The process for forming

  the hybridoma described above will now be described below.

after.

  Formation of a hybridoma capable of producing an anti-

  monoclonal body against bovine lactoferrin A human lactoferrin preparation having a purity of 98% is marketed by Sigma Co., but

  Bovine lactoferrin is not available in the

  commerce. Casein is therefore extracted from bovine colostrum by isoelectric precipitation and the resulting milk-yeast fraction is treated according to a conventional method [eg Johansson, B.G. et al .: Acta Chem. Scand.,

  23, 683, (1969) to obtain a lactofer-

  bovine with a purity of about 60%.

  Then a lactoferrin solution is prepared.

  vine (usually using phosphate buffered saline (PBS), pH 7.2, containing 0.15 M sodium chloride) and mixed with an equal amount

  of Freund's adjuvant to form an emulsion. We immunize

  three times, at two-week intervals, a

  mice (usually 6 to 8 weeks old)

  both this emulsion in the abdominal cavity. For this

  to do, one must use a highly pure form of lac-

bovine toferrin.

  To this end, it is desirable to increase the

  purity of bovine lactoferrin using an anti-

  serum against bovine immunoglobulins to remove immunoglobulins which are the main impurities

  present in the bovine lactoferrin fraction.

  If the purity of bovine lactoferrin is

  However, 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

  body against bovine lactoferrin is not brought to a

full level.

  There is no particular limitation in this

  the type of mouse used according to the present

  the invention. However, it is usually desirable

  to use BALB / c strain mice.

  According to the prior art, spleen lymphocytes

  for use in the formation of a hybridoma

  to produce a monoclonal antibody are usually

  lysely recovered from an excised mouse spleen 3 or 4 days after the final immunization with a specific antigen. In the case of bovine lactoferrin which is concerned by the present invention, the title of

  the antibody observed in the mouse does not reach a

  maximally 3 or 4 days after the final immunization, and the cells producing the antibody among the spleen lymphocytes are not sufficiently activated at this time. As a result, the rate of formation of a desired hybridoma, capable of producing a monoclonal antibody against

  lactoferrin, is of unsatisfactory weakness.

health.

  The applicant has found that the rate of training

  of the above hybridoma can be greatly increased by using spleen lymphocytes collected from the excised spleen from a mouse 6 or 7 days later.

  the final immunization with lactoferrin.

  Spleen lymphocytes (hereinafter referred to as cell

  spleen) collected from the mouse spleen as described above, are then fused with mouse myeloma cells (hereinafter referred to as

myeloma cells).

  There is no particular limitation regarding the type of myeloma cells used

  in the present invention. However, when

  to merge them with spleen cells obtained from BALB / c strain mice, it is preferable to use SP2 / 0Ag14 cells which do not secrete

  the K chain of IgG. The merger can be carried out se-

  in any conventional mode of operation. However, when

  SP2 / O-Ag14 cells are used as cell

  myeloma, it is essential that the melting time, including the addition of a fusion promoter (fusion inducer), mixing and dilution) be in the range

  from 5 to 15 minutes and preferably from 9 to 11 minutes.

  If the melting time is less than 5 minutes

  yours, the merger will be incomplete while if it is

  more than 15 minutes, the cells will die

  poisoning with polyethylene glycol used as a fusion promoter. In this regard, if the duration of merger

  is in the range of 9 to 11 minutes, the training rate

  colonies will approach 100%. When spleen cells from BALB / c strain mice immunized with bovine lactoferrin are fused with SP2 / O-Agl4 cells, the rate of colony formation

  (the melting rate) can be significantly increased by

  polyethylene glycol grade "gas chromatograph" (molecular weight 4000, Merck & Co., Inc.) in

  as a 50% merger promoter.

  When the fusion is complete, the cells

  can be treated conventionally.

  More precisely, the fused cells are dispersed

  in HT medium (Dulbecco MEM modified medium).

  with hypoxanthine, thymidine and 10%

  fetal calf rum), sprayed over a 96-hole microtiter plate and cultured at 37 ° C under an atmosphere of 5% carbon dioxide. AT

  from the following day, the selection of

  bridomas in HAT medium (Dulbecco-modified MEM medium containing hypoxanthine, aminopterin,

  thymidine and 10% fetal calf serum).

  As soon as the colonies have reached a sufficient size

  As a result, the hybridomas are screened according to

  the solid phase method. Then the hybridomas present

  both a positive reaction are cloned according to the method

dilution limit.

  According to one embodiment, the phasor method

  solid is carried out by causing the adsorption of a soluble antigen on a soft microwell at 96

  holes, treating the wells with serum albumin

  vine (BSA) to block portions on which

  the antigen is not adsorbed and the supernatants are

  in the wells to carry out the reaction with the antigen. Lors-

  the reaction is complete, the wells are washed

  and antibodies to antibodies

  biotinylated reagents are added as

  ondary. Following this, the wells are treated with avidin and fluorescein-labeled biotin to detect the desired antibody by producing fluorescence. The soluble antigen used for this purpose must be Bovine lactoferrin with as high purity as possible. The reason for this is that if the purity of bovine lactoferrin is low, the hybridomas for the impurity antigens also show a positive reaction and this makes it difficult to find a

  desired hybridoma producing a monoclonal antibody

be bovine lactoferrin.

  According to one embodiment, the method of

  the limit is implemented as follows:

  of 108 mouse thymus cells and 50

  Hybridoma cells in 10 ml of HT medium are sprayed on top of a 96-hole microtiter plate, so that one or more hybridoma cells are present in each hole and therefore single colonies of hybridoma are formed. Mouse thymus cells are added as load cells, since hybridoma cells can not grow at all.

low cell densities.

  The cloning process described above is

  three or more times to obtain a single hybridoma

  clone. Then the resulting hybridoma capable of producing a monoclonal antibody against bovine lactoferrin can be grown in the usual manner. More

  specifically, the hybridoma is injected into the abyssal cavity.

  mice and their ascites fluid is recovered.

  Father. Alternatively, the hybridoma is cultured in half

  suitable place and its supernatant is recovered. The Li-

  ascitic fluid or the recovered supernatant is then

  precipitated by ammonium sulphate precipitation and

  ion exchange matography to get the antibody

  monoclonal against bovine lactoferrin.

  According to one embodiment, the monoclonal antibody

  clonal against bovine lactoferrin can be prepared as follows: cells of the above-mentioned hybridoma are dispersed in phosphate buffered saline (PBS) and injected into the abdominal cavity of mice

  of BALB / c strain to which pristane (2,6,10,14-tetanus)

  tramethylpentadecane) was administered beforehand.

  After 7 to 10 days, their ascites fluid is collected

  clarified by centrifugation, diluted with PBS to give a protein concentration of the order of 10 to 12 mg / ml and then salted out with ammonium sulfate at 45% saturation. A solution of the precipitate fraction

  resultant is dialyzed and then purified by chromatogra-

ion exchange.

  Then, the monoclonal antibody against lactobacillus

  bovine ferrine thus obtained can be used in the

  separation and purification of bovine lactoferrin.

  This can be done by preparing an antibody affinity chromatography column according to the procedure described below and passing a solution of separated crude bovine lactoferrin from

  milk through the chromatography column of

  nity. Preparation of an affinity chromatography column The monoclonal antibody against lactoferrin

  bovine prepared in the manner described above is

  mixed with an equal amount of an insoluble support for use in affinity chromatography (eg

  Affigel-10 example; Bio-Rad Co.) and this mixture is

  at a low temperature to bind and fix the antibody to the support. After washing, the functional groups on the support to which the aforesaid antibody is not attached, are inactivated and the support is then washed. The resulting affinity gel is packed in a column of

  Suitable size to form a chromate column

  affinity graph. Raw bovine lactoferrin ob-

  held from the milk is dissolved in phosphate buffered saline (PBS) and the resulting solution is passed through the aforesaid column. Then the affinity gel inside the column is washed with PBS to remove the unadsorbed fraction, followed by a thorough wash with PBS containing 0.5 M sodium chloride and then a 0.15 solution. M sodium chloride. As a result, the adsorbed bovine lactoferrin is eluted with a buffer solution

  acetate, pH 2.7, containing 0.15M sodium chloride.

  The eluted fraction containing the bovine lactoferrin is adjusted to pH 7, dialyzed against deionized water and then lyophilized to obtain bovine lactoferrin having

a purity of 98% or more.

  The present invention is further illustrated by

detail by the following example.

Example

  By using an ion exchange resin, a fraction of bovine lactoferrin is separated from bovine colostrum according to the conventional method of Johnsson, B.G.

  coll .: Acta Chem. Scand., 23, 683, (1969) 7. This fraction

  Bovine lactoferrin has a purity of 60%.

  To the bovine lactoferrin fraction above,

  an antiserum is added against the immunoglobulins

  vines. Thus, the bovine immunoglobulins constituting the main impurities are eliminated to increase

  the purity of bovine lactoferrin up to about 90%.

  Bovine lactoferrin thus obtained is dissolved

  in phosphate buffered saline (PBS), pH 7.2, containing 0.15 M sodium chloride to give a concentration of 0.3%. This solution is mixed with an equal amount of Freund's complete adjuvant. (Difco) to form an emulsion. A BALB / c mouse, aged 6 to 8 weeks, is immunized by injecting the emulsion into its abdominal cavity. This immunization is repeated three times at 2 week intervals. Seven days after the final immunization, the spleen is excised from the mouse and the cells

  of spleen are collected from it. These

  The spleen cells are dispersed in DMEM medium (half

  Dulbecco-modified minimum essential site) and mixed with SP2 / 0-Ag14 mouse myeloma cells in a ratio of 2: 1. To this mixture is added a 50% solution of polyethylene glycol (gas chromatographic grade, MW 4000, Mechck & Co., Inc.) as a fusion promoter. So the fusion of the cells

  is completed in 10 minutes. The merged cells re-

  sultants are separated from polyethylene glycol by

  trifugation, dispersed in HT medium to give a cell density of 1 x 107 cells / ml or less, sprayed over 96-hole microtitre plates

  then grown at 37C under an atmosphere of 5% diode

  carbon dioxide. From the day after the beginning of the culture, (i.e., the first day), half of the medium is continuously exchanged for HAT medium. On the 17th day, the hybridomas are screened according to the

  solid phase method. It can be seen that 100% of hybrids

  domes form a colony and 8.3% of the hybridomas present

  a positive reaction for bovine lactoferrin.

  Then the hybridomas presenting a po-

  sitive are transferred to 24-hole microtiter plates and cloned as soon as their growth reaches a cell density of I x 105 cells / ml. Following

  of which, they are grown in HT medium for 2 seconds.

  and hybridomas forming a single colony in

  the well are subjected to secondary cloning.

  This process of cloning and screening is repeated

* three times. Thus, we obtain monoclones of a hy-

  bridome capable of producing an antibody against lac-

bovine toferrin.

  A resulting hybridoma culture capable of producing a monoclonal antibody against lactoferrin

  is maintained at the American Type Culture Collection.

  12301 Parklawn Drive, Rockville, Maryland 20852-

  1776 (U.S.A.) under deposit number ATCC HB 8852.

Claims (6)

claims   1. Hybridoma capable of producing a monoclonal antibody against bovine lactoferrin, which may be   to specifically combine with bovine lactoferrin,   characterized in that the hybridoma is obtained by fusing spleen lymphocytes from an immunized mouse   against bovine lactoferrin, with myeloid cells mouse lome.   Hybridoma according to claim 1, characterized   in that the spleen lymphocytes are collected from an excised mouse spleen 6 or 7 days later the final immunization.   3. Hybridoma according to claim 1 or 2,   characterized in that mouse myeloma cells are SP2 / O-Ag14 cells.   4. Method for forming a hybridoma capable of producing a monoclonal antibody against lactoferrin   bovine, characterized in that it comprises the steps of   able to use a solution of bovine lactoferrin   separated from the whey fraction of the milk; to immunity   mice with an emulsion formed in a mixture of   equal amounts of the lactofer-   rine and Freund's complete adjuvant; collecting spleen lymphocytes from the spleen of the mouse; fusing spleen lymphocytes with mouse myeloma cells and culturing the resulting fused cells; selecting the hybridomas formed in HAT medium; to screen the hybridomas selected according to the solid phase method to obtain a   hybridoma exhibiting a positive reaction; and to   hybridoma to a cloning process to obtain a monoclonal hybridoma.   5. Method according to claim 4, characterized   in that the solution of bovine lactoferrin is prepared   separating bovine lactoferrin from whey fraction of bovine colostrum by adding an antiserum against bovine immunoglobulin to lactoferrin   to fix and eliminate bovine immunoglobulins   and other impurities present therein, and to   wield the resulting purified bovine lactoferrin in phosphate buffered saline (PBS).   6. Process according to claim 4 or 5, characterized   characterized in that spleen lymphocytes collected from an excised mouse spleen 6 or 7 days after final immunization with bovine lactoferrin are used as spleen lymphocytes,   SP2 / O-Ag14 are used as myeloid cells.   mice, and these cells are fused for a period of 5 to 15 minutes and preferably 9 to 11 minutes.