CS221878B1 - Method of cultivation of hybride cell lines - Google Patents

Abstract

The invention relates to a method of cultivation hybridoma cell lines using production lines animals when irritating production animals prior to hybridoma inoculation preferably paraffin oil in a single dose of 10 to 50 Ail / g body weight production animal 5 to 20 days before inoculation

Description

The invention addresses the problem of culturing hybridoma cell lines while producing ascitic fluid containing monoclonal antibodies.

The concentration of many physiologically important substances, such as hormones or individual components of blood plasma, is increasingly used in human and veterinary medical diagnostics by an increasing number of immunological methods in which a specific antibody derived from the serum of the animal to which the desired substance is administered is central. The disadvantages of the antibodies prepared in this conventional manner are their heterogeneity and the inability to obtain identical antibodies from different individuals. These drawbacks eliminate the highly standard monoclonal antibodies that are produced by hybridomas, i.e., cells engineered artificially by so-called cell hybridization. In addition to innovation in medical diagnostics, the invention of a method for preparing monoclonal antibodies has opened new perspectives for the effective isolation of industrially important high molecular weight substances, as shown by the example of interferon purification developed in the UK. The hybridoma antibody production system, ie cellular hybrids whose hereditary basis has undergone some modification by human intervention, has the advantage over traditional animal antibody production that hybridomas retain genetic information about the specificity of the antibody synthesized, whereas each animal intended for antibody production must be repeatedly stimulated the respective antigen. The investment invested in constructing a hybridoma line providing the antibody of the desired properties will return many times because the hybridoma can for years produce an antibody of standard properties without further application of the antigen, the preparation of which is often expensive and labor intensive.

Monoclonal antibodies produced by B cell hybridoma cell lines are increasingly suited to the increasing demands of medical diagnosis. As opposed to conventional antibodies, they have a sharply defined specificity because they are chemically and functionally homogeneous products of a single cell line. Appropriate in vitro or in vivo propagation of the clone can yield practically unlimited amounts of these antibodies, thus ensuring a standard analysis run in national laboratories. Monoclonal antibodies will be produced at a lower cost, since the need for new immunizations of the test animals and the subsequent modification of the serum specificity will be eliminated.

B hybridoma cell lines result from the fusion of normal spleen lymphoid cells with myeloma cells, that is, from two parents of the B cell nature; myeloma cells carry the ability of unrestricted proliferation in vitro. The resulting hybrid cell line, the hybridoma, can be propagated in vitro in media suitable for lymphoid cell tissue cultures or in vivo in the peritoneal cavity of animals of the species of origin. The systems for the preparation of mouse and rat hybridomas are now well developed. When propagating hybridoma cells in the peritoneal cavity, monoclonal antibodies are released from the cells into the ascites fluid and recovered therefrom after sacrifice. Propagation in the peritoneal cavity has the advantage that the cell culture medium provides the organism in which the inoculated hybridoma cells live and there is no need for expensive in vitro culture media. Previously, a process of inducing myelomas in the periteneal cavity of mice has been used in which a susceptible strain of mice, the BALB / c strain, is induced by tumor-like myeloma growth by injection of paraffin oil or its components. In this process, the paraffin oil component of 2,6,10,14-tetramyl tylpentadecane has proven significantly more effective than unfractionated paraffin oil.

The process of inducing new myeloma always took several months. Our invention, in contrast to this long-term process, relates to the irritation of the peritoneal cavity of mice to prepare the biological terrain for attachment and intensive growth of the subsequently introduced hybridoma cells. The individual hybridomas behave quite differently to each other in in vivo growth, although analogously to myeloma cells. Indeed, the principle of hybridoma design determines that half of the properties of a hybridoma originate from a normal cell, among which there is considerable variability. Only the other half of the properties come from a cell of the myeloma nature. Thus, hybridomas cannot behave in the same way as myelomas and it is not possible to automatically assume the same growth requirements as they grow.

221876

Hybridoma cell inocula introduced into the peritoneal cavity without irritating substance in 60 to 80% of cases do not continue to proliferate and are destroyed by the organism. Therefore, prior to cell inoculation, an irritant substance is administered to the animals to adjust the physiological conditions in the peritoneal cavity so that the inoculum in all cases takes up, proliferates and stimulates the ascitic fluid ejection. The method of irritation is essential for the recovery of the monoclonal antibody. The hybridoma cells are inoculated into the peritoneum 4 to 6 weeks after the second injection of the irritant, with 0.5-1 ml of the irritant consumed per mouse. Until now, it has been known to use exclusively 2,6,10,14-tetramethylpentadecane as the irritant, which was generally preferred to paraffin oil without a comparative study. It was found by the inventors that the higher effect of 2,6,10,14-tetramethylpentadecane compared to the paraffin oil known from Eiyeloma induction is not evident in hybridoma culture in vivo.

The invention further provides a method for culturing hybridoma cell lines to produce ascites fluid containing monoclonal antibodies for immunodiagnostic purposes using production animals, wherein the irritation of the production animals is performed prior to inoculation of the hybridoma cells by irritating with paraffin oil preferably in a single dose of 10 to 50 Jul per gram body weight of production animal, 5 to 20 days before inoculation. A dose of 0.5 ml paraffin oil guarantees inoculum proliferation in 100% of cases, with ascitic fluid production and average antibody content being the same as previously used for more lengthy and costly procedures. At the same time, the presently proposed procedure significantly reduces the length of time the test animal is in the monoclonal antibody production cycle.

The invention and its effects are explained in more detail in the description of an embodiment below.

Example

Three groups of 20 mice weighing 25 to 30 g each were taken into the comparative experiment.

The first group was injected into peritoneum 2,6,1 0,14-tetramethylpentadecan at 0.5 ml, weeks before cell inoculation, the second group received paraffin oil at a dose of 0.5 ml I week before cell inoculation. The third control group received no irritant. The inoculum size in all groups was 4x10 6 cells. Animals of all groups were sacrificed 2 weeks after inoculation of the hybridoma cells. In the third non-irritant control group, poor cell growth was observed in 15% of the animals, and the other inoculum was discarded.

In both experimental groups, hybridoma cells were expanded and ascites fluid was collected.

In the experimental group receiving 2,6,10,14-tetramethylpentadecane, the mean volume of the ascites fluid was 4.4 ml and the average concentration of the monoclonal antibody was 5.3 mg / ml. In the paraffin oil treatment group, the average volume of the ascites fluid was 4.8 ml and the average concentration of the monoclonal antibody was 5.5 mg / ml.

Claims (1)

SUBJECT OF THE INVENTION A method for culturing hybridoma cell lines to produce ascites fluid containing monoclonal antibodies for immunodiagnostic purposes using production animals, wherein the irritation of the production animals is performed prior to inoculation of the hybridoma cells, wherein the irritation is performed with paraffin oil preferably in a single dose. up to 50 µl per gram body weight of the production animal 5 to 20 days prior to inoculation.