Sergey Borovikov, Aitbay Bulashev, Zhanbolat Suranshiev, Marat Koibagarov, Vladimir Kiyan

Biotechnology Scientific Research Institute of S.Seifullin Kazakh Agro Technical University, Astana, Kazakhstan

Receiving monoclonal antibodies to diagnostically valuable antigens of cattle’s bovine leucosis virus

Abstract

Three strains of hybrid cultivated cells stably synthesizing monoclonal antibodies to protein antigen of cattle’s bovine leucosis virus (BLV) with molecular mass of 24 kD and one strain to antigen with molecular mass of 51 kD were received with the method of hybridoma technology. Characteristics of monoclonal antibodies were studied and total immunochemical characteristics were given which allows concluding that received antibodies can be useful at the development of immunological tests for diagnosing cattle’s leucosis.

Introduction

Diagnostics of cattle’s leucosis is based on clinical, serological, hematological and pathology anatomy data. However long incubation period, variety of diseases display and deleted symptomatic, low antibodies’ titers in individual trials of serum or patterns received from patterns of pathological material complicate to diagnose with these methods JOHNSON and KANEENE, 1991). Therefore high sensible immunochemical methods of diagnostics such as enzyme linked immune sorbent assay (ELISA) (DOLTZ and MORENO, 1999; MOLLOT et al., 1990) are the most preferable nowadays. Different variants of this test are the most usable, reliable and recommended to international epizootic offices (OIE.CHAPTER 2.4.11. ENZOOTIC BOVINE LEUCOSIS, 2000).

The main important factor defining sensitivity and specificity method of analysis are antibodies. The method of hybridoma technology allows getting stable preparations of specific high affinity monoclonal antibodies which effectively are used in diagnostic systems for identification of different antigens and other biologically active substances (BRUCK et al., 1982 f, BRUCK et al., 1982b, JAFARI JOZANI et al., 2008).

Receiving hybridoma strains producing monoclonal antibodies to diagnostically significant antigens (p24 and gp51) of cattle’s bovine leucosis virus is the aim of the given work.

Materials and methods

Recombinant antigens p24 and gp51 of cattle’s bovine leucosis virus were used in the work.

Two groups of mice of Balb/c line (by analogs method) three heads in each group were selected for immunization. From them the first group was immunized with gp51 antigen and the second with p24 antigen. Mice were immunized with purified virus antigens during two weeks. For that reason intraperitoneal introduction of 100 mkg of each antigen in 0,1 ml partial Fraind’s adjuvant (SIGMA) was injected. On the 7^th, 11^th, 12^th, 13^th days of immunization animals were injected with 100 mkg antigen in buffered physiological solution pH 7,2 – 7,4. Increasing level of antibodies was checked in indirect variant of immune – enzyme analysis against initial antigen. Hybridization of cells with reinjected mice myeloma line X63Ag8.653 with splenocities of immune mice was conducted by standard method using 50% solution of PEG 4000 (Fluka) (OI and HERZENBERG, 1980).

Hybrid cells’ cloning was carried out with the method of limited breeding (GDING,1980).

Hybridoma cells in partial growth medium were injected into mice’s abdominal cavity (quantity of cells 2õ10⁶) of BALB/c line for whom 2,6,10,14 tetrametilpentadekan (Sigma) in 0,5 ml was injected per head before 7-10 days. Ascitic fluid (3-10 ml per mouse) is got on the 10^th-14^th days after cells inoculation. Immunoglobulin fraction was singled out from ascitic fluid with sulphate ammonium salting out.

Electrophoresis was transmitted in 10% polyacrilamid gel with presence of sodium dodecylsulfat (SDS) at apparatus for vertical electrophoresis. Definition of antigen determinant to which Mab has got, was conducted with western blot method. Transferring of divided antigens was implemented to nitrocellulose membrane (Millipore) with the help of apparatus for immunoblotting Semi blot (10x10sm).

Definition of Mab isotypes was conducted in immunodiffusion reaction with the help of specific antiserum to different classes of mice’s immunoglobulin (IgG Fc Subclass 1, IgG Fc Subclass 2 a, IgG Fc Subclass 2 b, IgG Fc Subclass 2 c, IgG Fc Subclass 3, IgM). The results of reaction were taken into account by the presence of precipitation stripes between hollows with tested antigens and specific antiserum.

Indirect and “sandwich” variants of hard phase immune – enzyme analysis were conducted at 96 hollow plane tables for immunological reactions (Nunc, Denmark) in standard variants. Positive reaction is characterized with painting substrate solution into yellow – brown colour. The results of ELISA were taken into account with the help of spectrophotometer with vertical light flow at 492 nm wave’s length.

Results

Taking blood from immunize mice were fulfilled in 3 days after the last immunization and it was investigated in indirect variant of ELISA with starting antigen on containing specific antibodies. The results of research have shown that antigens used for immunization fully fit for stimulation of tested animals’ immune system. Antibodies’ titers in blood serum reached sense from 1:6400 till 1:102400. It points out the active induction of B- lymphocytes producing antibodies of the given specific character.

After 4 days of the last injection with the method of cervical dislocation animals with the highest titers of antibodies in ELISA were used for getting immune splenocytes. Hybridization of immune B lymphocytes and myeloma cells were conducted by standard method.

After 14 days of hybridization control of amalgamated cells growth was carried out under the inverted microscope. The growth of hybrid cells was observed in 64 (8%) from 768 hollows used for scatter.

Hybridoma testing for specific antibodies production was begun from the moment of growth environment yellowness and hybrid cells have taken more than 30% of hollows’ surface. Strains of cultural medium were selected in the volume of 1,1 ml and were investigated for antibodies’ content in ELISA.

The results of the first hybrid clone testing have shown the presence of specific antibodies against starting antigens of bovine leucosis virus in 4 hybrid strains. All hybridoma strains were subjected to clone with the method of limitative dilution. The results of clone have showed homogeneity of selected sub clones as 94% of them have preserved synthesis of specific immunoglobulin. Antibodies‘ titers of cultural subclone fluid was in range 1:16 – 1:32 where antibodies’ coupling in ELISA was discovered. Strains of the most active hybridoma sub clones were defined after completing cells’ cloning.

Ascitic fluid was used for injection of immunoglobulin received in the result of inter abdomen cells’ injection to mice.

Comprehensive studying of hybridoma strains’ immunochemical properties which defining the perspective of their further usage is the most important stage of the work in creation of hybrid strains producents (manufacturer) of monoclonal antibodies.

Isotypes of monoclonal antibodies synthesized with hybrid cells’ strains are turned out to be different: Mab of two sorts belonged to the class IgM and two others belonged to IgG1 and IgG3.

One of the main properties of monoclonal antibodies is such index as affinity which can be measured by quantity with the help of defining coupling constant. Coupling constant of taken monoclonal antibodies as regards to the used antigens makes up from 1,9õ10^-8Ì äî 2,5õ10^-8Ì.

Western blot with starting antigens used for immunization was conducted for more detail epitope analysis of monoclonal antibodies. Positive reaction of antibodies of 3B8 strain as regards to antigen with molecular mass 51kD was registered. Immunoglobulin of other strains displayed great activity as regards to protein antigen with molecular mass of 24 kD.

The whole characteristics of immunochemical properties of monoclonal antibodies synthesized with hybridoma’s strains were taken after some complex research (table 1).

Table 1 – Characteristics of hybridoma strains

Starin’s name	Coupling constant	Mab isotype	Productivity in vitro, mg/ml	Productivity in vitro, mg/ml	Specificity
3B8	1,9õ10^-8Ì	IgM	0,06	2	51kD
4G7B4	1,6õ10^-8Ì	IgM	0,03	2	24kD
2F9B11	2,5õ10^-8Ì	IgG3	0,03	1	24kD
1D9E7	2,2õ10^-8Ì	IgG1	0,125	2	24kD

As it is seen from the table the received strains have high productivity (maximum synthesis Mab in vitro – 2 mg/ml) which is important index in groundwork of immunoglobulin on industry scales. Synthesized monoclonal antibodies have rather high indexes of coupling constant. It will allow providing reliable coupling of immunoglobulin with specific antigens. Specificity regards to homological and heterological organisms is one of the main characteristics of monoclonal antibodies defining scientific and practical value for the further application. Ascitic fluid of sub clones with antigens of homological and heterological viruses was tested for checking specificity of immunoglobulin. Specificity of immunoglobulin taken to antigens of leucosis virus turned out to be high and at the experiment they reacted only with starting antigen.

Thus 3 strains of hybrid cultivated cells stably synthesizing Mab to protein antigen BLV with molecular mass of 24 kD and 1 strain to antigen with molecular mass 51 kD were received. It is known that in many cases anti virus antibodies are formed against glycated antigen of virus surface gp51 and against inner protein p24. According to this received antibodies can be useful at the development of immunochemical tests for diagnosing of bovine leucosis virus.

References

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