SU1740415A1 - Strain of hybrid cultured cells of animals mus musculus l as a producer of monoclonal antibodies to the pseudomonas pseudomalei antibodies, which do not react with malleus infection pathogenic organism of the nearest relationship - Google Patents

Abstract

Uses: biotechnologists, immunologists. SUMMARY OF THE INVENTION: A strain is obtained by fusing with polyethylene glycol of mouse myeloma cells RZ-X63-A.8.653 with Balb / c mouse lymphocytes. The strain was deposited in the Specialized Collection of Transplanted Somatic Cells of Vertebrates of the Institute of Cytology of the Academy of Sciences of the USSR under the number VSKK (II) 388D. The strain produces monoclonal antibodies (µA) type a IgM, the titer of µA in the culture fluid - 1:40 - 1: 160, in ascites fluid - 1: 104 - 1: 105. ICA interact with typical strains of Pseudomonas pseudomalleei and do not interact with typical strains of Pseudomonas mallei. The stability of antibody production is maintained for 28 passages without loss of activity. MCA is used to identify the pathogen melioidosis. 1 tab. (L

Description

The invention relates to hybridoma technology and relates to the production of a hybrid clone-producer of monoclonal antibodies to the species-specific antigen Pseudomonas pseudomallei.

Until recently, for the diagnosis, identification and differentiation of P. pseudomallei, absorbed monospecific animal sera were used to the pathogen melioidosis (basic object), but because of the presence of common antigens, even after absorption, they gave cross-reactions with the closely related pathogen of glanders (P. mallei).

The purpose of the invention is the production of hybrid cultured Mus musculus L cells producing specific monoclonal antibodies to an antigen.

Pseudomonas pseudomallei that do not react with the closely related causative agent of glanders.

A new strain obtained using traditional methods of hybridoma technology. The peculiarity of its creation is that for hybridization (using PEG) with mouse myeloma cells (RH-X63-A8.653), lymphocytes of mice, initially immunized with intraperitoneal corpuscular antigen P.pseudomallei (1,110 m), were taken. t.) and for the second time stimulated by the same antigen in culture in vitro (1 10 mt / ml). The strain of hybrid cells, SCC (II) 388D, was deposited and stored in the specialized collection of transplanted somatic cells of the vertebrates of the Institute of Cytology of the Academy of Sciences of the USSR

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The BCCC (II) 388D strain (the author’s name is MMR.rm 2 (clone 45 C4 B2) has the following characteristics. Cultivation conditions.

1. vitro, in the form of a stationary suspension: temperature 37 ° С, atmosphere with 5% С02 and 98% humidity, Pskov – Khema – RPMI medium (1: 1: 2) with ethanolamine (up to 300 / l M) or medium RPMI-1640 containing 10% fetal cow serum, HEPES (10 mM), 1-glutamine (2 mM) without antibiotics or with up to 100 units / ml penicillin and streptomycin added. Cells (transparent, rounded sometimes in the form of conglomerates that disintegrate during pipetting) are scattered after 3-4 days with a multiplicity of sowing 1: 3 - 1:10 (optimal sowing dose: 150-200 thousand cells in 1 ml of medium).

2. Passages in vivo in BALB / c mice, pretreated with sterile vaseline oil or with a pre-strap (0.5 ml intraperitoneally in 7-14 days), are administered by intraperitoneal injection of 3-5 106 cells of a hybrid clone. On days 7–28, ascites develops in the abdominal cavity of animals. In the culture fluid (in the in vitro system) and in the ascitic fluid (in the in vivo system), monoclonal antibodies to P.pseudomallei antigen are contained. The titer of specific immunoglobulins in the culture fluid and ELISA (enzyme-linked immunosorbent assay) 1:40 - 1: 160, in ascitic fluid - 1: 104 - 1: 105. In the immunofluorescence test (in the interaction of specific antibodies with bacteria), salted monoclonal antibodies react with the antigen at a dilution of 1: 20000.

Properties of antibodies produced by the hybrid strain.

Monoclonal antibodies are classified as JgM. The stability of antibody production is maintained for 22 passages in the in vitro culture and for 28 passages under cultivation with the change of host (in vivo - in vitro in vivo) (observation period).

In comparative trials in ELISA and immunofluorescence reactions (direct and indirect), monoclonal antibodies produced by hybrid cells interact with the P.pseudomallei antigen and do not interact with the antigens of the closely related causative agent of glanders (P.mallei), i.e. have a strict species specificity to the pathogen melioidosis. This was also confirmed by comparative testing of antibodies with sets of typical museum strains: the antibodies reacted with all tested typical strains.

P.pseudomallei and did not react with any

from tested typical P.mallei strains.

Use of the product received

a new strain of hybrid cells - monoclonal antibodies to the species-specific antigen P.pseudomallei,

Monoclonal antibodies are obtained either from culture fluid (when cultured in vitro) or from

0 ascitic fluid (when cultivated hybridomas in vivo). In order to demonstrate their selective (with respect to the pathogen melioidosis) specificity, an enzyme immunoassay method, direct and indirect immunofluorescence is used. Linked immunosorbent assay. As an antigen on the solid phase for comparative analysis in ELISA, water-salt extracts from suspensions are used.

0 inactivated bacteria (two-day cultures of either P.mallei or P.pseudomallei, typical strains with a complete set of antigens inherent in each species), subjected to sonication and

5 centrifugation clarification. In order to sensitize the plates, pre-selected optimal doses of total antigens (20 µg / ml in carbonate-bicarbonate buffer, pH 9.6), sensitivity assay - overnight at 4 ° C are used.

The table contains comparative testing data in ELISA of monoclonal antibodies produced by hybrid clones on different antigens (total

5 antigens of the causative agent of glanders or melioidosis),

The table shows that the antibodies of the hybrid strain interacted with the antigen of the pathogen melioidosis and did not

0 reacted with antigens of the glanders pathogen, Only in low dilutions of ascitic fluids 1:20 - 1:80 a possible positive reaction at the background level, apparently, due to non-specific binding

5 serum antibodies. In high dilutions, in the presence of a positive reaction with a specific antigen, there were no cross-reactions with glanders antigens. There were no cross-reactions when testing the antibodies of the hybrid clone from the culture supernatant (when grown in vitro in hybridomas). In contrast, monoclonal antibodies to LPS of the pathogen melioidosis reacted with total

5 antibodies of the causative agent of glanders and the causative agent of melioidosis, demonstrating the fact that LPS is a common antigen for both pathogens. Taken as a negative control, monoclonal antibodies to diphtheria toxoid did not react with either

one of the antigens. Thus, in the ELISA using the proposed antibodies, differentiation of these pathogens is possible.

Indirect immunofluorescence method

Slices of bacteria suspensions of pathogens of melioidosis and glanders are smeared on slides in two rows, drops of dilution series of monoclonal antibodies are fixed and applied to them. In the control, known reacting (+) or non-reacting (-) antibodies. Incubation is performed in a humid chamber, washed with saline, labeled fluorescent antibodies to mouse immunoglobulins are added at working dilution, incubated, washed, dried, and viewed under immersion. The smears with the presence of microbial fluorescence are considered positive. When testing monoclinal antibodies of the proposed hybrid strain, it was found that they do not give fluorescence when tested on corpuscles of the glanders pathogen and give a distinct fluorescence of corpuscles of the melioidosis exciter (diluted to 1: 20000).

Direct immunofluorescence method

Monoclonal antibodies are commonly conjugated with fluorescein isothiocyanate by the conventional method and used in the direct immunofluorescence test with the pathogen melioidosis. It has been established that when conjugated with fluorescein isothiocyanate, the specific activity of monoclonal antibodies does not decrease. When analyzing a series of museum strains of both pathogens in direct and indirect immunofluorescence tests, it was found

that the proposed antibodies react with the pathogens of melioidosis and do not react with the glanders pathogens, i.e. have a pronounced species specificity.

The high specificity of monoclonal antibodies produced by the proposed cell strain (species specificity) allows in one reaction (immunofluorescence reaction, ELISA) to provide early diagnosis, identification of the causative agent of meliondosis and its differentiation from the closely related causative agent of glanders. While the previously absorbed monospecific serums to the pathogen melioidosis (basic object) give cross-reactions with the glanders causative agent, the proposed antibodies have species specificity. These antibodies (the product of the proposed hybrid strain) can be considered as a highly active and specific ingredient for the formulation of indirect immunofluorescence reactions and as a raw material for the manufacture of fluorescent immunoglobulins for the formulation of direct immunofluorescence.

Thus, the new strain provides a more effective drug suitable for identifying the causative agent of a particularly dangerous infection, meliodosis.

Claims (1)

Invention Formula The strain of hybrid cultured animal cells Mus musculus L., BCKK (II) No. 388D is a producer of monoclinal antibodies to the Pseudomonas pseudomallei antigen, which do not react with the closely related causative agent of glanders. The results of testing in the ELISA preparations of monoclonal antibodies to the disblock of 8SCK (II) 368 L (the supernatant from the culture of hybrid cells or a mixture of ascitic fluids from mice with hybridomas &