Immunosorbent to obtain monospecific antisera to transthyretin person

 

(57) Abstract:

Usage: in medicine, when creating the immune test systems to determine the transthyretin (TTR) of a person in biological fluids. The essence of the invention: immunosorbant to obtain monospecific antisera to TTR person includes fibrinoliticeski active blood plasma of a person with total protein content 155,8 mg/ml, free from transthyretin, ovalbumin at a concentration of 65-90 mg/ml and as cross-linking agent is glutaric aldehyde at a final concentration of 0.7-0.8 vol.%. Immunosorbent allows researchers to simultaneously remove all non-specific antibodies from the antisera to TTR person, reduces the adsorption time and can significantly reduce the cost of production of diagnosticum to TTR person. 2 Il., table 2.

The invention relates to medicine and can be used in the medical industry when creating the immune test systems for determination of transthyretin (TTP) person in biological fluids.

Study the content of the TTP in the serum of patients will be used to assess the degree of failure and monitoring the effectiveness of clinical nutrition, monitoring the development of premature infants. The change in the patients with thermal, traumatic brain injury, some therapeutic diseases.

The main stage of the creation of a diagnostic test system is to obtain monospecific antisera. After immunization of animals with purified drug transthyretin get antisera containing, typically as a mixture of antibodies to proteins of human blood plasma. In this regard, there is a need to remove nonspecific antibodies from such antisera to TTP person. The adsorption of antisera is recommended for adequate sorbents containing proteins that interact with the impurity antibodies [1,2,3] However, universal sorbent does not exist.

The objective of the invention is the creation of a universal immunosorbent assay, increasing the specific activity of the antisera to the TTP person.

To create a universal sorbent must have a biological environment of man, containing all the proteins of blood plasma, except for the TTP. In the process of adsorption of these proteins will interact with non-specific antibodies and, consequently, to increase the activity of the antisera to the TTP person.

Unique biological environment of the person we encountered. Ustanovki range of proteins of blood plasma (PL. 1). This specific transfusion medium is widely used in clinical practice as a thrombolytic tools [4]

The process of "exhaustion" of antisera most technologically advanced in the presence of solid immunosorbent assay, so it was used a crosslinking agent and a filler ovalbumin.

The problem is solved by mixing PFAP and ovalbumin to the concentration of total protein 75-110 mg/ml, using a cross-linking agent is glutaraldehyde at a final concentration of 0.7-0.8. by washing the obtained immunosorbent in buffer solutions at different pH.

Immunosorbent prepared as follows: to PPAP with a certain concentration of protein added to ovalbumin concentration 75-110 mg/ml of total protein. To this mixture is added 2.5% solution of glutaraldehyde to a final concentration of 0.7-0.8. dropwise, with constant stirring. After three hours at room temperature, a gel is formed, which is ground using a homogenizer and washed various buffer solutions.

"Exhaustion" of the antisera to the TTP person provide in a centrifugal tube mix anticigarette to TTP containing nonspecific antibodies and policendarlehen anticigarette filtered and subjected to immunochemical study.

Example.

Cooking immunosorbent:

To 50 ml PHAP (pH 7.0) with the concentration of total protein of 10.2 mg/ml (total protein pool was 510 mg) added 3500 mg of ovalbumin powder and mix thoroughly. After complete dissolution of the protein, dropwise, with constant stirring to the mixture is added 15 ml of 2.5% solution of glutaraldehyde. For 3 h at room temperature, a gel is formed, which is ground in a homogenizer and alternately washed in 0.2 M phosphate buffer (pH 7.3) and 0.1 M GLYCANS (pH of 2.8). The crushed gel (immunosorbent) store in a bottle with 0.02 sodium azide at +4-6oC until use.

Adsorption of antisera to TTP person:

In the centrifuge tube, mix 5 ml of the antisera to the TTP man (series 8-664/2 from the source specific titer of 1:10) and 5 ml immunosorbent assay. The mixture is stirred at Shuttle for 35 min at room temperature. Adsorbed anticigarette separated from the solid adsorbent by filtration and subjected to immunochemical analysis. Title adsorbed antisera was 1:4, it is revealed antibodies only to the TTP person.

Checking the activity of the hard immunosorbent was held with history is recipitation in the gel on Ouchterlony and through immunoelectrophoresis (Fig. 1,2). As a reference antisera were used serum against TTP "Sevac" (Czechoslovakia), and antigen TTP whole serum of human blood (a mixture of samples from 50 donors). The titer obtained monospecific antisera to the TTP was on average 1:6 (PL. 2).

Immunodiffusion analysis of antisera after adsorption showed that all series contain only antibodies to the TTP person and standard antigen TTP person ("Sigma", USA). Immunoprecipitate proved identical antigenic structures TTP person. All adsorbed antisera was completely absent nonspecific antibodies.

The efficiency of adsorption of non-specific antibodies further investigated through immunoelectrophoresis and cross-immunoelectrophoresis (Fig. 2). The titer of specific antibodies in adsorbed antisera to TTP ranged from 1:4 to 1:8.

Thus, the obtained immunosorbent allows researchers to simultaneously remove all non-specific antibodies from the antisera to the TTP person, its use reduces the adsorption time and can significantly reduce the cost of production of diagnosticum to TTP person. Note that the obtained immunosorbent can be repeatedly and the 1968, S. 294.

2. Immunological methods. Under the editorship of Primes, Moscow, "Medicine", 1987, S. 472.

3. The antibodies. Methods. Edited by Katie D. Moscow, "Mir", 1991, 2 h, 382.

4. C. B. Grips. "Drugs fibrinolytic and antiproteinase steps from plasma suddenly dead people". Summary of the doctoral thesis. M. 1984, 48 S.

Immunosorbent to obtain monospecific antisera to transthyretin person, including leaching fibrinoloticheskaya active blood plasma of a person with total protein content of 15 to 5.8 mg/ml, free from transthyretin, ovalbumin at a concentration of 65 to 90 mg/ml and as cross-linking agent is glutaric aldehyde at a final concentration of 0.7 to 0.8.

 

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