Method for preparing affinity surface based on chemically cross- linked protein a

FIELD: immunology, proteins.

SUBSTANCE: invention proposes a method for preparing affinity surfaces based on chemically cross-linked staphylococcus protein A. Method involves applying a layer of staphylococcus protein A on hydrophobic surface followed by chemical cross-linkage of protein A. Also, method involves additional applying specific antibodies on the prepared affinity surface with a layer of chemically cross-linked staphylococcus protein A. This method provides preparing the affinity surface eliciting the enhanced strength that can be separated from a backing if necessary and with retaining its integrity. Also, the flat and uniform surface of layer of crossed-linked staphylococcus protein A provides low level of interferences in carrying out study by different methods of microscopy. Invention can be used in investigation of biological objects by different methods, among them by method of atomic-force microscopy. Invention can be used in immunological researches of biological objects.

EFFECT: improved preparing method.

3 dwg, 3 ex

 

The invention relates to the field of environmental protection, medical and microbiological industry. The invention consists in the preparation of affine surfaces designed for specific immobilization of biological objects by applying onto a hydrophobic surface layer of staphylococcal protein a, chemical internal firmware of this layer, and application to the surface of antibodies specific for a particular ligand. Properties of complex protein a - antibody such that in the terms of use affine surfaces in liquids containing ligand) antibody on the surface layer of protein And focused active sites in the liquid that provides efficient capture ligand.

Affine surfaces are widely used for highly sensitive detection of the presence of high-molecular compounds and biological objects of submicron size in an environment with highly specific allocation of various substances from the mixture, the concentration (affinity chromatography, plasmapheresis). In analytical and medical purposes are also particles with affine surface. Despite the diversity of methods using affine surfaces, the number of molecular mechanisms specificity, and appropriate ways of organizing affin what's surfaces is small. Variants of molecular interactions, providing specificity, the most frequently used interaction of antigen - antibody. Recently, widespread use of specific hybridization of the DNA fragments or RNA, but the molecular mechanism is only applicable for the detection of DNA or RNA. At the same time, the interaction of the antigen - antibody more versatile. Currently tested and widely used methods of preparative obtain specific antibodies against a wide spectrum of antigens.

The number of ways of obtaining an affine surfaces based on antibodies, i.e. fixing antibodies or their active fragments on the surface, is also insignificant. The greatest strength provides covalent fixation, but may significantly limit the mobility of the active sites of the antibodies, which leads to a significant drop in the efficiency of the capture ligand. This disadvantage is largely overcome by the use of flexible molecules intermediaries between the antibody and substrate. In addition, covalent fixation involves carrying out a chemical reaction, which may adversely affect the ability of the active sites of molecules for specific binding.

Some additional facilities provides approx is out for fixing antibody avidin-bioteknologi complex, one of the components of which (usually - avidin) covalently attached to the substrate, and the other (Biotin) antibody. In a solution of avidin and Biotin spontaneously form a complex; in the "biotinylated" antibody is fixed on a coated Avidya surface. When using this scheme, you receive a opportunity to store for a long time ready affine surface (with consequent risk of its destruction during storage), and to prepare it immediately before use, since the formation of the avidin-bioteknologi complex does not require complex special conditions and for a long time. The disadvantage of this method can be attributed to the need of biotinylate antibodies, which increases the cost and requires special procedures.

One of the most convenient ways of fixing antibody on the substrate is pinning layer protein A. Nature of protein structure And is such that it is able to bind the Fc - fragment of any IgG antibodies, which gives the opportunity to prepare affine surface based on IgG antibodies without special handling.

A typical example of use for analytical purposes is fixed on a solid inert carrier (glass fibers) agent capable of specific binding (namely covalently attached antibodies), is the tsya patent [1]. In biochemical tests are also widely used as carriers of antibodies polymers, such as agarose [2, 3]. An example of the use of affine surfaces based polynucleotides can serve as a patent [4]. In some cases, the detection of antigens is convenient to use a non-static surface or the media, which are fillers biochemical columns, and particles with affine surface [5, 6].

An example of the use of affine surfaces in medicine, in the columns for purification of blood, can serve as a patent [7], where it is used to combat atherosclerosis, leukemia, autoimmune diseases. To remove antibodies from the blood needed to deal with some autoimmune diseases, can be used in protein And covalently attached to a silicon matrix placed in biochemical column [8, 9]. In the process of using these columns, it was found that a similar therapeutic effect have and particles washed off with them and fall in the blood plasma. Was subsequently developed a special way of cooking has a therapeutic effect of particles of the chemically bound protein And [10]. There is also known a method of immobilization of protein a, in one embodiment, adsorbed on particles of activated carbon placed in collodial is embrane, and the other covalently attached to the particles of nylon, polystyrene, methacrylate [11]. This system is also used for purification of blood plasma.

The essence of the invention is to develop a simple and effective method of preparing stable affine surfaces of the substrates on the basis of protein a, which is then adsorbed antibodies. To achieve this goal in the first phase on the surface of the hydrophobic substrate was formed a layer of protein A. Protein a is a membrane protein Staphilococcus aureus. Protein molecule And has a hydrophobic areas, which under normal conditions of existence of the organism is immersed in its membrane, and the hydrophilic areas, oriented to the environment, capable of binding the Fc fragment of antibodies, which is the basis of natural biological protection Staphilococcus aureus.

In the artificial conditions of the solution selected protein And the hydrophobic surface under certain conditions, the layer is formed, in which the hydrophobic areas adjacent to the substrate, and a hydrophilic, as in the case of microorganism, converted into a solution and are able to effectively link placed in a solution of antibodies. This active group of antibodies are also oriented in the solution, i.e. the optimal to capture antigens. This explains the fact that on surfaces with high the hydrophobicity, such as coal [11], are able to form effective affine layer of protein a and antibodies. Covalent anchoring layer protein And the substrate is not always possible to achieve the correct orientation of the molecules in the layer, which affects the efficiency of the subsequent binding of antibodies.

The disadvantage of the adsorbed layer of protein And is low strength. To overcome this drawback without losing the correct orientation of the protein molecules in the layer we offer after adsorption layer on the substrate to produce its internal chemical firmware using a crosslinking agent, without chemical prishivki to the substrate. Then, on the thus prepared layer of adsorbed antibodies; the resulting affine surface is used for carrying out the immunological assays and can be used in other above-listed fields.

From methods using adsorbed on the substrate protein And the proposed method is characterized by the presence of chemical treatments firmware layer protein. From methods using covalent binding of the protein with the media, including those used in the patent [11] is the fact that the protein is not sewn with the media, and used the internal firmware of protein A. In the method described in [10], internal chemical firmware protein a is used, but the protein is used in the form is Uspenie isolated particles, not fixed on the flat surface of the carrier. In our proposed method, the hydrophobic carrier is not only the backbone of affine surface, but also ensures the correct orientation of the protein molecules And the most effective for the immobilization of antibodies.

Despite the absence of covalent bonds with the substrate, the film bound protein And very firmly on it, because when you try tear strength of intermolecular bonding, van-der-Waals forces are cooperative. If necessary, the film can still be separated from the substrate. While it retains unlike the case with the covalent anchoring to the substrate its integrity and can be subjected to additional research (for example, using a method of transmission electron microscopy).

The method was developed primarily for use in studies of biological objects mounted on affine surfaces using the atomic force microscopy (AFM). Figure 1 shows obtained using this method, the image layer staphylococcal protein a bound glutaraldehyde, on an atomically smooth graphite surface. Dimensions in microns. The surface layer is smooth and uniform, which ensures a low noise level when conducting research. Figure 2 shows obtained using skaniruesh the th electron microscope image of a film of protein And, separated from the substrate. Noteworthy that separated the film retains its integrity and, if necessary, can be investigated by other means. Figure 3 shows the bacteria E.coli, mounted on prepared using the proposed method affine surface, the carrier-specific data pertaining to bacteria antibodies.

The technical result of the development of this method is the creation of affine surfaces used for detecting event-specific sorption using modern methods of microscopy, as well as allowing you to apply for this and other physical methods of detection, which are based on surface effects. Universalization of the cooking surface allows you to explore the same object using different methods of detection.

Example 1

1. Protein a (lyophilized derived from Staphylococcus aureus) dissolved in distilled water to a concentration of 1 mg/ml

2. The solution was applied to svezheokleennuju the surface of a plate of graphite in the amount of 3 μl. The sample was then dried in air (under traction) at room temperature until completely dry within 2-4 hours.

3. On the spot protein to cause 1% solution of glutaraldehyde in distilled water with a volume of 3 µl; sample incubated in a humid atmosphere (in a Cup Perimete with a sheet of filter paper, moistened with water) for 2 hours.

4. The plate is washed by placing on the surface of the active layer down to 5 minutes.

5. After removal of residual water, but without additional drying on the surface of the stain is applied 0.3 M buffer solution of Tris-HCl, pH=8.8 per 1 hour.

6. After washing, similar to item 4 on the spot put 3 μl of a solution of antibodies (polyclonal antibodies rabbit serum against Esherichia coli PCs-12) for 30 minutes, then spend washing.

7. On the spot put 3 ál of primary immobilising object (product Esherichia coli PCs-12, the concentration of 108cells/ml) for 30 minutes. After washing, the sample is dried under traction for 2 hours and passed for research. The image obtained using an atomic force microscope (Digital Instruments, 3A) is shown in figure 3. Observed binding of bacteria Esherichia coli PCs-12 with the surface of the graphite, covered with a layer of protein a and antibodies specific for these bacteria.

Example 2

1. Protein a (lyophilized derived from Staphylococcus aureus) dissolved in distilled water to a concentration of 1 mg/ml

2. The solution was applied to svezheokleennuju the surface of a plate of graphite in the amount of 3 μl. The sample was then dried in air (under traction) at room temperature until completely dry within 2-4 hours.

3. On the spot protein to cause 1% solution is laterolog aldehyde in distilled water, volume 3 ál; sample incubated in a humid atmosphere (in a Petri dish together with a sheet of filter paper, moistened with water) for 2 hours.

4. The plate is washed by placing on the surface of the active layer down to 5 minutes.

5. After removal of residual water, but without additional drying on the surface of the spots are 0.5 M solution of glycine, pH=8.5-by 1 hour.

6. After washing, the stain is applied 3 μl of a solution of antibodies (monoclonal antibodies specific dispute vaccine strain of Bacillus anthracis STI, concentration 0.1 mg/ml) for 30 minutes, then spend washing.

7. On the spot put 3 ál of primary immobilising object (drug dispute WHEREIN the concentration of 109spores/ml) for 30 minutes; after washing, the sample is dried under traction for 2 hours and transferred to research. Observed binding dispute STI with affine surface more than 100 pieces in the area of 10×10 ám.

Example 3

1. Protein a (lyophilized derived from Staphylococcus aureus) dissolved in distilled water to a concentration of 1 mg/ml

2. The solution was applied to svezheokleennuju the surface of a plate of graphite in the amount of 3 μl. The sample was then dried in air (under traction) at room temperature until completely dry within 2-4 hours.

3. On the spot protein cause a 0.1% solution dimethylaniline the ATA, pH=8,0 volume of 3 μl. The sample is incubated in a humid atmosphere for 2 hours.

4. The plate is washed by placing on the surface of the active layer down to 5 minutes.

5. After removal of residual water, but without additional drying on the surface of the stain is applied 0.05% solution of monoethanolamine, pH=8,1 1 hour.

6. After washing, the stain is applied 3 μl of a solution of antibodies (monoclonal antibodies against Legionella bacteria macdedi, the concentration of 0.2 mg/ml) for 30 minutes, then washed again.

7. Put 3 ál of primary immobilising object (the product of the bacteria Legionella macdedi, the concentration is 5·107cells/ml) for 30 minutes; after washing, the sample is dried under traction for 2 hours and passed for research. Observed binding of the bacteria Legionella macdedi surface that carries antibodies that are specific in relation to these bacteria, more than 30 bacteria on the area of 25×25 microns.

The list of used literature

1. U.S. patent 5861319.

2. U.S. patent 6379908.

3. U.S. patent 6376204.

4. U.S. patent 6221581.

5. U.S. patent 5898005.

6. U.S. patent 6121056.

7. U.S. patent 5753227.

8. U.S. patent 4614513.

9. U.S. patent 4681870.

10. U.S. patent 6447777.

11. U.S. patent 5091091.

1. The method of obtaining the affine surfaces, providing for applying a layer of protein And the hydrophobic surface and subsequent chemical stitching is the protein A.

2. The method according to claim 1, characterized in that it further cause antibodies to bound protein A.



 

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