The allocation method c3component of the human complement

 

(57) Abstract:

The invention relates to medicine, in particular to biochemistry and immunochemistry, and can be used in the diagnosis of diseases associated with activation of the complement system. The invention consists in that an alcohol residue IV - I Stake precipitated 16% poly (ethylene glycol) m m 4000 - 6000. The resulting precipitate emit C3component of complement, using ion-exchange chromatography on DEAE-Toarea 1 and gelfiltration on sephacryl S-200. The proposed method of purification of C3component of the human complement of sediment IV - I in the line has advantages over the prototype, because the method does not require expensive reagents, raw materials, simple in implementation. Advantages of the invention is to simplify and reduce the cost allocation method C3component of complement, waste disposal industrial fractionation of plasma.

The invention relates to medicine, in particular to biochemistry and immunochemistry, and can be used in the diagnosis of diseases associated with activation of the complement system: diseases of immune complexes, infectious diseases, respiratory distress syndrome of adults, hereditary defeo3component of the human complement. For example, the widely used method of selection3component of the complement of the plasma, proposed by R. A. Harrison and R. J. Lachmann in 1979 , involving pre-treatment of fresh plasma inhibitors (EDTA, sodium azide, phenylmethylsulfonyl) to prevent activation of proteolytic enzymes in blood plasma and starting a cascade of reactions in activation of the complement system with subsequent fractionation of plasma sodium hydrosulphate to 20% and presidenial precipitate formed 11% of sodium sulfate. The supernatant from the second deposition is divided into pseudoglobulin and euglobulin faction in dialysis low ionic strength and slightly acidic pH. WITH3component of complement extracted from sediment euglobulin by ion-exchange chromatography on DEAE-cellulose and chromatography on hydroxyl-Apatite. Finish the selection WITH3component of the complement dialysis3-containing fractions against solution inhibitors (EDTA, NaN3), ultrafiltration.

The prototype of obtaining high-purity WITH3component of the complement is the method described Tach. B. F. and Prahly. W. (1976). It includes: a step fractionation of fresh plasma is matography on L-lysine-sepharose 4B to remove plasminogen, ion-exchange chromatography on DEAE-cellulose, gel filtration on sepharose CL-6B, chromatography on hydroxyapatite) with control hemolytic activity at each stage. The method is sequential, time-consuming, it is used of expensive reagents and sorbents for chromatography.

The aim of the present invention is to simplify and reduce the cost allocation method WITH3component of the human complement, which is especially important for diagnostic products, and recycling of waste industrial fractionation of plasma.

The objective is achieved by the fact that3component of complement allocate only 3 main stages, using as source3component waste sludge IV-I at Stake. According to Cohnetal. (1946) in the sediment IV-I, obtained by the fractionation of blood plasma in the cold, contains components of complement.

The first stage of the selection WITH3from the precipitate IY-I is the precipitation of 20% sediment IV-I, dissolved in 0.1 M K-R, pH 7,1-7,3, 10 mm EDTA, 0.5 M NaCl poly(ethylene glycol) 4000-6000. 32% PEG 4000-6000 add a 20% solution of sediment IV-I to a final concentration of 16% PEG 4000-6000 under stirring. The formed precipitate was separated by centrifugation for 20 min at 6000 rpm, 4varaut in 0.01 M K-R, pH 7.5, 10 mm EDTA and chromatographic on the column with DEAE-Tooreal I, equilibrated with the same buffer, elution of bound peroxidase protein, using a linear concentration gradient of NaCl 0-0,3 M Identification of fractions containing3conduct immunohistochemistry. Further concentrated using ultrafiltration membrane PM-30 component WITH3additionally purified by gel-filtration on sephacryl S-200 in 0.01 M K-R, pH 7.5, 10 mm EDTA, 0.15 M NaCl, 0.02% Of NaN3.

The proposed method of cleaning WITH3component of the complement of the bargain sediment IV-I in the line has advantages over the prototype, because the method does not require expensive reagents, simple, has only 3 main stages: I - precipitation PEG 4000-6000;

2 DEAE-chromatography;

3 - gel-filtering.

Cleared WITH3component of complement can be used: 1) as the immunogen for hyperimmunization animals in order to obtain monospecific anti-C3precipitating serum; 2) to remove unwanted anti-C3antibodies of the polyvalent sera; 3) to determine the titers of anti-C3precipitating serum by the method of REED Mancini.

THE ALLOCATION METHOD C3COMPONENT of COMPLEMENT CHE the relevant clearing of sediment on DEAE-aminoalkanoic and gel-filtration, characterized in that is used as raw material sediment IV - I fractionation of blood plasma at Stake, before processing the PEG it is dissolved in a buffer solution of high ionic strength at neutral pH and gel-filtration is carried out with the help of sephacryl S - 200.

 

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