CS230288B1 - Method of preparing mono-clonal antibodies - Google Patents

Description

The invention provides a method for preparing monoclonal antibodies to tick-borne encephalitis.

The first monoclonal antibodies were produced in 1975 [O. Kohler, C. Milstein: Nature 256, 495, (1975)]. In the preparation of hydridomas to tick-borne encephalitido (fusion of myeloma cells with the spleen cells of an immunized animal using polyethylene glycol), antibody-producing clones are obtained. After cell reclamation, final propagation is made in the peritoneal cavity of histocompatible animals to which Freund's adjuvant, reisp. Land (2, 6, 10, 14 - tetramethylpentadecane, Koch Light Lab. Ltd. Anglia) to create ascitic fluid [M. Novak, M. Gresikova, M. Sekey, G. Russ, J. Zikan, M. Pospisil, F. Ciampor: Acta virol., (1983)].

SUMMARY OF THE INVENTION The present invention relates to a method for the preparation of monoclonal antibodies, characterized in that inactivated Staphylococcus aureus is injected into the peritoneal cavity of an imbred line of mice, inoculated into a buccal broth, incubated for 18 hours at 37 ° C. 60 ° C for 30 minutes, held at 4 ° C for 18 hours; the material is again inactivated by heat, scentri2 is fused and sediment is used in the experiments.

An advantage of the proposed method of preparing monoclonal antibodies against tick-borne encephalitis is that the antibodies specifically react in a hemagglutination-inhibition assay. The monoclonal antibodies obtained make it possible to differentiate tick-borne encephalitis virus from theBO virus in the family Togaviridae. The invention allows a culture of hybridoma cells in the peritoneal cavity of histocompatible mice. The advantage of this method also lies in the high concentration of monoclonal antibodies.

Example

Inoculate 1000 ml of a buccal broth with Staphylococcus aureus; the infected broth is incubated for 18 hours at 37 ° C. After inactivation of Staphylococcus aureus at 60 ° C for 30 minutes, it is allowed to stand for 18 hours at 4 ° C. It is inactivated again at 60 ° C for 30 minutes. After centrifugation, 2 g of sediment is obtained. The sediment is resuspended in 30 ml of broth. One millillter preparation contains 60,000,000 cells. The BALB / C mouse imbred line is used in experiments. Mice are given 1.5 ml of inactivated Staphylococcus aureus 54 days prior to immunization. Mice are immunized with tick-borne encephalitis virus, a Skalica strain that is stored in Cs. Collection of Arboviruses of the Institute of Virology of the Slovak Academy of Sciences in Bratislava under the designation Clgl and number 53 Skalica, propagated on smokers embryonic cells and purified by differential centrifugation. The first dose of virus administered intraperitoneally contains 5120 hemagglutinating units; the second dose given after 35 days contains 320 haemagglutination units. Hybridoma lines (myeloma line PgNS1 / 1 Ag 4-1) and spleen lymphoid cells that produce Moinoclonal Antibodies in vitro are detected using a tick-borne encephalitis haemagglutination inhibition assay, Skalica strain. After cloning and recloning, hybridoma lines are obtained with a sustained ability to produce monoclonal antibodies. Further, only those capable of producing moinoclonal antibodies are maintained. In order to obtain a large number of monoclonal antibodies, myeloma hybrid cells are administered to BALB / C mice that are inactivated with Staphylococcus pyogenes aureus 54 days prior to treatment. AsciClone number

vitriol

The acetone treated antibody containing the monoclonal antibody has a hemagglutination-inhibition titer of 1: 162.240 for clone # 1. 9 and 1:80 for clone no. 16 (Table 1j. Prepared monoclonal antibodies are characterized in that they contain identical class M immunoglobulins (early antibodies) whose heavy chain isotype is μ and the light chain type 1 is κ. The neutralization index after the addition of anti IgM antibodies was 2.4 µ clone # 9 and 2.1 for clone # 16.

Monoclonal antibodies are of great importance in medical diagnostics as well as in basic research. Product standardization and differential diagnostics are possible.

T a b u 1 'k a 1

Results of hemagglutination-inhibition and neutralization test with monoclonal antibodies to tick-borne encephalitis prepared in ascitic fluid of BALB / C mice using inactivated Staphylococcus aureus

Titer of haemagglutination-inhibiting antibodies Neutralization index with monoclonal antibody antigen

Antibodies treated by neutralizing with telsacetone. and 2-mercapto were added with amtitoethanol -IgM antibody

162240

80

2.4

2.1

Claims (1)

230288 prior to immunization. Mice are immunized with tick-borne encephalitis virus, a strain of Scalpel that is deposited in Cs. collections of arboviruses at the Institute of Virology of the Slovak Academy of Sciences in Bratislava under the designation Clgl and number 53 Skalica, propagated on smokers by embryonic cells and purified by differential centrifugation. The first dose of virus administered intraperitoneally contains 5120 hemagglutinating units; the second dose administered after 35 days contains 320 hemagglutinating units. Hybridoma lines (PgNS1 / 1 Ag 4-1 myeloma lineage) and spleen lymphoid cells that produce moinoclonal antibodies in vitro are detected using the hemaglucinating-inhibition assay of the aintigene encephalitis, Skalica strain. After cloning and recloning, hybridoma lineages with a sustained ability to produce monoclonal antibodies are obtained. Furthermore, lenties that are capable of producing monoclonal antibodies are maintained. In order to obtain a large quantum of monoclonal antibodies, myeloma hybrid cells are administered to BALB / C soaps, which are administered 54 days inactivated by Staphylococcus pyogenes aureus. Asci-Clone number Skalica Antibodies treated with acetone-tonic fluid containing monoclonal antibodies have hemagglutinin-inhibitory tether 1: 162.240 for clone # 9 and 1: 80 for clone 16 (Table 1). The prepared monoclonal antibodies isa are characterized in that they contain identical class M immunoglobulins (early antibodies) whose heavy chain isotype is µ and the 1 'light refractory κ type. The neutralization index after addition of anti-IgM antibodies was 2.4 for clone # 9 and 2.1 for clone # 16. Monoclonal antibodies are of great importance in both medical diagnostics and basic research. It enables product standardization and differential diagnosis. TABLE 1 Results of the hemagglutination-inhibiting aneutralization assay with monoclonal antibodies to tick-borne encephalitis prepared in ascites fluid BALB / C mice using inactivated Staphylococcus aureus Titer of hemagglutinin-inhibiting antibodies Neutralization index with monoclonal anti-antigen neutralizing tels-acetone. and 2-mercapto were added to the aintiethanol-IgM antibody 9 162.240 16 80 0 2.4 10 2.1 SUBSTANCES A method for producing monoclonal antibodies characterized in that an in-line mouse is administered to the peritoneal cavity by an imbred line. activated Staphylococcus aureus, which was inoculated into the buffer broth, incubated at 37 ° C for 18 hours, then inactivated at 60 ° C for 30 minutes, maintained for 18 hours at 4 ° C, the material heat inactivation, centrifugation, and use of experiments.