METHOD OF PRODUCING AND DETERMINING SPECIFIC ACTIVITY OF MACROPHAGE ACTIVATION FACTOR GcMAF Russian patent published in 2024 - IPC C12Q1/00 G01N33/48 

FIELD: biotechnology.

SUBSTANCE: described is a method of producing and determining specific activity of the macrophage activation factor GcMAF. Method is based on extraction, conversion and determination of specific activity. At that, macrophage activation factor GcMAF is recovered and converted on solid carrier Sephacril/25-OH D3—Sepharose with covalently bonded to it vitamin D3, hydroxylated in 25th position, under conditions of affine sorption of the macrophage activation factor on the resin particles. For its validation using: specific antibodies and matrix ribonucleic acid (mRNA), synthesis of which is activated by macrophage activation factor GcMAF in peritoneal macrophages (PMP) of C57BL/6 mice; a set of specific primers and a real-time polymerase chain reaction (PCR) procedure. Raw material used is a mixture of donor plasma, diluted in half with physiological saline or phosphate-buffered saline, and standardization of the macrophage activation factor GcMAF is carried out by isolating the factor on Sephacril/25-OH D3 substrate—Sepharose with covalently attached to it vitamin D3 hydroxylated in 25th position. Further, DBP is converted to a macrophage activation factor GcMAF in a Sephacril/25-OH D3 substrate bound state. Activity of each release of the preparation of the cell system of PMP of mice of the C57BL/6 line is determined and the ability of DBP and GcMAF to induce synthesis of PMP mRNA of four cytokines—inflammation mediators TNF-α and IL-1β and anti-inflammatory cytokines TGF-β and IL-10, wherein during conversion, purified enzymes β-galactosidase and sialidase present in the conversion medium are used, and the ability of PMP to synthesize cytokines is determined using a real-time PCR procedure, where the specific primers used are primers for pro-inflammatory cytokines TNF-α and IL-1β and anti-inflammatory cytokines TGF-β and IL-10. Invention can be used to obtain and determine the specific activity of the macrophage activation factor GcMAF, which is a Gc-protein and a vitamin D carrier, which neutralizes G-actin released during cell lysis.

EFFECT: invention widens the field of application and increases efficiency.

1 cl, 8 dwg