Method for obtaining liposomal-immunoperoxidase conjugate

FIELD: biotechnologies.

SUBSTANCE: liposomes are used as a matrix for activated ferment - horseradish peroxidase. To 5 mg of horseradish peroxidase oxygenated with a periodate method there added is 1 ml of liposome suspension in 0.01 M solution of carbonate-bicarbonate buffer at pH 9.5, and subjected to ultrasonic treatment during 1 min. Then it is incubated for 1 hour; immobilised with immunoglobulins in concentration of 5 mg during 2 hours at the temperature of 22±4°C; stabilised with 5 mg of sodium borane with further gel-chromatographic cleaning.

EFFECT: invention allows obtaining liposomal-immunoperoxidase conjugate for indication of infectious agents in an immunoenzymometric analysis and increasing service life of a preparation up to 6 years.

1 tbl, 3 ex

 

The invention relates to biotechnology and can be used in the production of immune-peroxidase conjugates for indication of infectious diseases in the enzyme-linked immunosorbent assay (ELISA).

Enzymes - specific protein substances with the ability to accelerate chemical reactions with subjectyou structure and exposed to the negative influence of temperature, buffer changes, the concentration of hydrogen ions and so the shelf Life is limited and varies from 6 months (for liquid medications, at a storage temperature of minus 4°C) up to 1 year (for lyophilised, at a storage temperature of 4-6°C).

A method of obtaining enzyme conjugate, comprising the activation of the enzyme and immobilization with immunoglobulins [1].

A method of obtaining enzyme conjugate, comprising lyophilization immunoperoxidase conjugate in solution of bovine serum albumin (BSA) and store at 4°C [2].

The disadvantage of the methods is the limited shelf life - up to 1 year.

The purpose of the invention to increase the shelf life immunoperoxidase conjugate with the preservation of its physico-chemical and immunological parameters by fitting the parameters of the immobilization of the enzyme to the matrix.

The technical result of the invention is achieved by the fact that the quality of the matrix are used liposomes (with adjustable composition, size, surface properties, non-toxicity, does not inactivate the enzyme, allowing to operate with high activity and so on), immobilized activated horseradish peroxidase associated with immunoglobulins are purified from unbound components.

You need to design and select the method of immobilization and to determine the influence of the latter on the enzyme. It is known that the enzyme is able to perform the role of a specific catalyst in compliance with mainly two conditions. First, the enzyme must be in the right conformation. Secondly, he should be able to change its conformation in the process of catalysis.

As a matrix, you need to choose one that would not hinder the penetration of the substrate and not "chained" enzyme, depriving him of mobility necessary to perform its inherent function.

Taking into account the above factors and taking into account the properties of liposomes, it should be noted that the most appropriate matrix for the enzyme is horseradish peroxidase and the task at hand - getting immobilized immunoperoxidase conjugate are liposomes.

The nature of the preparation of immobilized enzyme depends on the nature containing the enzyme phase. Is there a way to attach the enzyme using electrostatically who or other non-covalent binding mechanisms. On the other hand, the enzyme does not need to be attached to the carrier, and can be kept inside it. While the media forms around the enzyme setuptool matrix, the cells of which are so small that a molecule of the enzyme can be released from the network, but at the same time large enough for the penetration of low molecular weight substrates [3]. One of the variants of this method lies in using a double phospholipid layer as the enzyme phase. In this case, the enzyme can be in aqueous solution, surrounded by a phospholipid barrier, or be dissolved in the hydrophobic part of the double layer.

Technology of production of liposomal-immunoperoxidase conjugate consisted of several stages: obtaining liposomes; the inclusion of the enzyme, immobilization with immunoglobulins; purification from unbound components, lyophilization and control. Liposomes obtained using chloroform-ethanol mixture, and the rate of diffusion of cations through the membrane increases. The raw material used gomogenizirovannykh the brain of a pig. After extraction was performed separation of lipids by adding 20% of the volume of the extract of 0.9% solution of sodium chloride. After incubation for 2-3 h lipids were separated. The value of the quantity of lipids ranged from 30 to 50 mg/ml of the lipid Composition was determined by means of thin layer of HRO is ecografia on the plates "Silufol" [4].

Lipids obtained as described above, consisted of phosphatidylcholine, phosphatidylserine, phosphatidylinositol and cholesterol. Then spent the evaporation of the extract lipids on a rotary evaporator to form a film of lipids on the walls of the flask. This step is very important due to the fact that the film lipids must be thin enough, otherwise undesired inclusion of a significant amount of dry phospholipid in a system of concentric bilayers, as a closed membrane will effectively prevent further access of water and salt. Then he washed the dried lipids with chloroform (based on 50 mg of lipids 3.5 ml of chloroform), was stirred flask to dissolve the precipitate, contributed 1.5 ml of 0.9% solution of sodium chloride, and chloroform was removed in a rotary evaporator. Determination of the size of liposomes was performed at the electron microscope and was calculated by the formula. The size of the resulting liposomes was 100-200 nm.

When receiving liposomal-immunoperoxidase conjugate initially conducted research in terms of the inclusion of the enzyme in liposomes. The most appropriate method was the inclusion of pre-activated horseradish peroxidase: (5±0,1) mg of horseradish peroxidase was dissolved in 1 ml of a 0.3 M solution of sodium carbonate acid was added to 0.025 ml of 0.32% solution of formalin. The mixture was mixed on a mixer in the course is e (30±5) minutes at a temperature of (22±4)°C and added 1 ml of a 0.04 M solution of periodate sodium, continuing to stir for (30±5) minutes at a temperature of (22±4)°C. Further contributed to 1.0 ml of a 0.16 M solution of ethylene glycol (to reduce surface tension), gently stirring in a period of (60±5) minutes at a temperature of (22±4)°C. At the end of the procedure, the drug is activated peroxidase should be transparent, odorless, greenish-brown color. The activated peroxidase was transferred into a dialysis bag and were dialyzed against 1 l of 0.01 M carbonate-bicarbonate buffer (CBS), pH of 9.5, for (19±1) h on the stirrer at a temperature of 2-8°C in the refrigeration chamber. Thus, the activation of the enzyme periodate oxidation, to form aldehyde groups.

Further, the step of receiving liposomal complex-immunoperoxidase conjugate was as follows. To 5 mg of oxidized periodate method peroxidase was added 1 ml of a suspension of liposomes in 0.01 M CBS pH of 9.5. The suspension was subjected to ultrasonic treatment (44 kHz) for 1 min, then incubated 1 h To liposomes immobilized horseradish peroxidase, was added tularemia immunoglobulins (Ig G) in an amount of from 2.5 to 10 mg in 3 ml of 0.01 M CBS pH of 9.5. Immobilization of immunoglobulins with activated horseradish peroxidase was carried out at a temperature of (22±4)°C, stirring at Shuttle for 2 h, stabilized 5 mg of borohydride in the refrigerator at a temperature of 4-6°C. the Probability is about, this peroxidase was included in the membrane of liposomes, and the aldehyde groups of the carbohydrate part of the enzyme interacted with the amino groups of immunoglobulins.

For purification of the conjugate from unbound enzyme used gel chromatography. The conjugate was applied on the column, 100×12 mm with Sephadex G-100, equilibrated with 0.1 M solution of the FSB pH of 7.2. This resulted in the fractionation of the mixture of molecules into zones depending on their size. Therefore, the first peak came liposomal-immunoperoxidase conjugate, then the unbound antibodies and the enzyme. The elution was performed in the same buffer, and fractions were collected in 2.0 ml and explored in the enzyme-linked immunosorbent assay (ELISA).

In the result set, which is essential for obtaining a quality product is the activation of the enzyme at the optimum time of immobilization 2 h and the protein concentration of immunoglobulins 5 mg/ml

To increase the shelf life of the medication was dispensed in ampoules, pre-adding 1% BSA, froze and liofilizirovanny for (18±2) h to a final temperature of 25°C. In a final product was controlled by the physico-chemical (solubility, color, transparency, loss in weight on drying) and immunological (activity, specificity, sensitivity) properties after drying and during storage is ment for 6 years (time of observation). The sensitivity and specificity of the conjugate was determined by ELISA with homologous and heterologous strains by the method of Dark M.F., Adams A.N. [5].

The table presents the results of physico-chemical properties and sensitivity of the drugs. Developed preparations meet the requirements of the regulations applicable to diagnostic drugs, and their stability is superior to traditional enzyme conjugates 6 times (observation period).

Thus, as a result of research developed the manufacturing technology of immobilized enzyme preparation that greatly increases its stability. This is due to the fact that the immobilization effectively prevents the harmful effects of environment on enzyme preparation, he becomes a prisoner in the matrix (liposomes), i.e. protected, in the absence of physical and catalytic restrictions, the matrix does not prevent the penetration of the substrate due to efficient method of obtaining liposomes, activate the enzyme and immobilization of antibodies, temperature selection, time parameters, holding gentle cleaning from unbound components.

The opportunity for practical application of the proposed method is illustrated by examples of its specific implementation using the receiving entirety of the claimed features.

Example 1. Getting the plague liposomal-immunoperoxidase conjugate and its control in ELISA

Pre-spent activated horseradish peroxidase: (5±0,1) mg of horseradish peroxidase was dissolved in 1 ml of a 0.3 M solution of sodium carbonic acid, was added to 0.025 ml of 0.32% solution of formalin. The mixture was stirred in a stirrer for (30±5) minutes at a temperature of (22±4)°C and added 1 ml of a 0.04 M solution of periodate sodium, continuing to stir for (30±5) minutes at a temperature of (22±4)°C. Further contributed to 1.0 ml of a 0.16 M solution of ethylene glycol, gently stirring in a period of (60±5) minutes at a temperature of (22±4)°C. the Activated peroxidase was transferred into a dialysis bag and were dialyzed against 1 l of 0.01 M solution of CBS pH of 9.5 for (19±1) h on the stirrer at a temperature of 2-8°C in the refrigeration chamber.

Obtaining complex liposomally-immunoperoxidase conjugate was carried out as follows: 5 mg of oxidized periodate method peroxidase was added 1 ml of a suspension of liposomes in a 0.01 M solution CBS pH of 9.5. The suspension was subjected to ultrasonic treatment for 1 min, then incubated 1 h To liposomes immobilized horseradish peroxidase, was added plague immunoglobulins (Ig G) in an amount of 5 mg per 3 ml of 0.01 M CBS pH of 9.5. Immobilization of immunoglobulins with activated horseradish peroxidase was carried out at a temperature of (22±4)°C, stirring at Shuttle for 2 h,stabilized 5 mg sodium borohydride in the refrigerator at a temperature of 4-6°C for 2 hours For purification of the conjugate from unbound enzyme used gel chromatography. The conjugate was applied on a column with Sephadex G-100, equilibrated with 0.1 M solution of the FSB pH of 7.2. This resulted in the fractionation of the mixture of molecules into zones depending on their size. In the first peak came conjugate, then the unbound antibodies and the enzyme. The elution was performed with the same buffer solution. The activity was tested in ELISA. The drug was dispensed in ampoules, pre-adding to the finished product 1% BSA, froze and liofilizirovanny for (18±2) h to a final temperature of 25°C. In a final product was controlled by the physico-chemical and immunological properties after drying and during storage. The sensitivity of the conjugate in ELISA with homologous strains was 1×104M.K./ml, in the absence of cross-reactions with heterologous strains. The shelf life is 6 years (observation period).

Example 2. Getting tularemia liposomal-immunoperoxidase conjugate and its control in ELISA

Was obtained analogously to example 1, except that instead of plague used tularensis antibodies. The sensitivity of the conjugate in ELISA with homologous strains was 5×104M.K./ml, in the absence of cross-reactions with heterologous strains. The shelf life is 6 years (the period of observation is possible).

Example 3. Getting brucellosis liposomal-immunoperoxidase conjugate and its control in ELISA

Was obtained analogously to example 1, except that instead of plague used Brucella antibodies. The sensitivity of the conjugate in ELISA with homologous strains was 1×105M.K./ml, in the absence of cross-reactions with heterologous strains. The shelf life is 6 years (observation period).

Sources of information

1. Nakane, P.K. Buffer-labelled antibody - a new method of conjugation / P.K.Nakane, A.Kawaoi // J. Histochem. Cytochem. - 1974. - Vol.22. - P.506-508.

2. RF patent №2276362, publ. 10.05.2006, "Method of detecting the virus Crimean-Congo hemorrhagic fever (KGL) in biological and field", the authors Vasilenko NF, Efremenko VI, Tyumentseva I.S. and other bull. No. 13.

3. Trevin M. Immobilized enzymes. - M.: Mir, 1983. - P.23.

4. Cats M. Technique of lipid. M.: Mir, 1975. - C-82.

5. Clark M.F., Adams A.N. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant virus // J. Gen. Virol. - 1977. - V.36, No. 3. - P.475-480.

The method of obtaining liposomal-immunoperoxidase conjugate for indication of infectious diseases in immune-enzyme analysis, characterized by the fact that as a matrix for the activated enzyme is horseradish peroxidase - use liposomes to 5 mg of oxidized periodate method of horseradish peroxidase was added 1 ml of suspension lipo is ω 0.01 M solution of carbonate-bicarbonate buffer at pH of 9.5, subjected to ultrasonic treatment for 1 min, incubated for 1 h, immobilized with immunoglobulin at a concentration of 5 mg for 2 hours at a temperature of 22±4°C, stabilized 5 mg of sodium borohydride and subsequent gel chromatographic purification.



 

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