The method of determining the concentration of the main protein heat shock 70 kda

 

The invention relates to medicine, namely to laboratory diagnosis. The method consists in the immobilization agent that interacts with HSP70 on the surface of the wells of 96-hole tablet for enzyme immunoassay, the washing of the wells with buffer, introduction into the wells of the studied sample or standard and incubation, washing of the wells, introduction into the wells of polyclonal rabbit antibodies to HSP70 and incubation, washing of the wells, introduction into the wells of the antibody that interacts with rabbit antibodies conjugated with an enzyme and incubation, washing of the wells, introduction into the wells of the substrate of the enzyme and Chromogen, incubation, spectrophotometric evaluation of developed color and correlate it with data calibration. As the immobilized agent that interacts with HSP70, use derived ATP covalently associated in a complex with HMDA with egg albumin. The samples and standards add sodium salt of ATP to 10 mm, and also contribute to the clipboard magnesium ions to a final concentration of 10 mm at all stages. The method has high sensitivity and accuracy. 3 Il.

The invention relates to the field of biology the LCA heat shock 70 kDa (HSP70) in tissue samples of animals and humans and biological liquids of an organism.

There is a method of determining the concentration of HSP70 [1], which consists in conducting the assay on 96-well tablets by immobilization of rabbit polyclonal antibodies to HSP70 with subsequent washing of the wells and making the sample with the addition of purified and biotinylated HSP70, incubation, following washing and making avidin conjugated with the enzyme horseradish peroxidase incubation and making after another washing of the substrate of horseradish peroxidase - hydrogen peroxide and Chromogen. Developing color is measured spectrophotometrically. The colour intensity is inversely proportional to the number nebutiniausias of HSP70 in the sample. The sensitivity of the method is only 40 ng/ml.

Closest to the claimed method is [2], in which the concentration of HSP70 is performed by ELISA on 96-well tablets by the interaction of immobilized monoclonal antibodies to HSP70 with the antigen contained in the entered into the hole of the sample during incubation and subsequent washing of the wells, the recognition of the captured antigen recognition rabbit polyclonal antibody made in the wells and wash the wells, Vienna provides a quantitative assessment of antigen in the sample due to the development of color painting, proportional to the amount of bound HSP70. The colour intensity is measured spectrophotometrically and compared with the calibration curve. The sensitivity of the method is also low at approximately 20 ng/ml in Addition, this method requires a pair of antibodies that interact with different parts of the molecule HSP70. The receipt of such pairs of time-consuming due to the nature of this protein.

The objective of the proposed method is the determination of the concentration of HSP70 in the tissue samples of animals and humans and biological fluids with high sensitivity and eliminates the need to obtain a second antibody to the molecule of HSP70 by using properties of HSP70 is peculiar to interact with immobilized adenosine triphosphate (ATP).

This objective is achieved in that on the surface of the wells of 96-hole tablet for enzyme immunoassay immobilized complexes of 8-(6-aminohexyl)-amino-adenosine-5’-triphosphate (GMD-ATP) (Fig.1) obtained by the known method [3], conjugated with molecules of egg albumin (Fig.2), the wells are washed with buffer, they make the test sample or standard, to which is added the sodium salt of ATP to a concentration of 10 mm, and the content and bacia, the wells are washed, they make antibodies which are reactive with rabbit antibodies conjugated with an enzyme, and incubated content, the wells are washed, they are the substrate of the enzyme and Chromogen, the contents and incubated produce spectrophotometric evaluation of the developed color with the subsequent assignment of this calibration data.

Features that distinguish the invention from the prototype are replaced immobilized in the wells of 96-hole PCB antibodies to HSP70 contingent of ATP, which has a higher affinity for HSP70, adding to the sample of the sodium salt of ATP to 10 mm, causing the dissociation of complexes of HSP70 with other proteins, including other HSP70 molecules, and, thus, demaskirovanie its antigenic determinants that contribute to a more complete interaction of HSP70 with recognizing rabbit polyclonal antibodies, as well as the entry in the buffer of magnesium ions to a final concentration of 10 mm at all stages, what is required for interaction with HSP70 ATP.

Graphic materials.

1) the structural formula of HMDA-ATP (Fig.1);

2) the conjugation of HMDA-ATP with egg albumin (Fig.2);

3) schematic illustration of consistently interacting elements of the complex (Fig.3).

connected to the molecules of the egg albumin through a hetero-bifunctional linker Succinimidyl-4(p-maleimidomethyl)-butyrate (SMPB), allows you to bind the amino group of HMDA-ATP with sulfopropyl molecules of cysteine that is part of egg albumin (Fig.2). Prepare a solution of the obtained complexes in buffer Tris Hcl pH 7.5, containing 140 mm sodium chloride to a final concentration of egg albumin, 10 μg/ml In wells of 96-well plate to enzyme immunoassay make 100 μl of a solution. During 60-minute incubation at 37°C is the immobilization of protein molecules with attached molecules derived ATP on the surface of the hole. Next is three times washing of the wells with 300 ál of buffer containing 0.2% detergent tween-20. The same buffer with 0.2% detergent tween-20, 140 mm sodium chloride and 10 mm magnesium chloride is used at all subsequent stages. Prepare dilution of purified HSP70 to build further calibration curve. The concentration of HSP70 in dilutions are 1 µg/ml, 250 ng/ml, 50 ng/ml, 5 ng/ml, 1 ng/ml, 500 PG/ml 250 PG/ml In the first well is added 100 μl of buffer solution not containing HSP70, as a negative control. All standard cultivation are applied in two holes each to control the reproducibility of the result. In the remaining wells are analyzed samples. In the hole at this stage is entered as is hivatala at room temperature, is three times washing of the wells with 300 ál of buffer. The following reagent, placed in the wells are 100 µl of a solution of rabbit polyclonal antibodies against HSP70. Hour incubation at room temperature and terminated by washing, after which the wells are to be paid the solution of the second antibody directed against rabbit and conjugated with the enzyme horseradish peroxidase. After an hour incubation at room temperature is used to wash holes and is made of 100 μl of the substrate of horseradish peroxidase - hydrogen peroxide and the Chromogen is of orthophenylphenol, in citrate buffer pH 4.5. After 30 minutes of incubation, the enzymatic reaction is stopped by adding 50 µl 0,19 M sulfuric acid to each well. The colour intensity is determined spectrophotometrically using reader 96-well plates at a wavelength of 450 nm. Largest acquisitions for known concentrations of HSP70, purified product which was made in the wells simultaneously with the test samples, the gage curve. The desired concentration of HSP70 in the samples is determined using this curve. Schematic representation of consistently interacting elements of the complex is shown in Fig.3. T the tissues of animals and humans and biological fluids with sensitivity on the order of a higher - up to 250 PG/ml, while the use of a derivative of ATP as an agent that binds HSP70, eliminates the need for antibodies to the alternative portion of the molecule HSP70. In addition, the use of antibodies to other known proteins that are able to make ATP, can dramatically broaden the scope of application of the proposed method.

Sources of information

1. Margulis C. A., Nacharov P. V., Tsvetkova O. I., M. Welsh, Kinev, A. V. The Characterization and Use of Different Antibodies Against the Major Hsp70 Heat Shock Protein Family for the Development of an Immunoassay. Electrophoresis. 1991, Sep., 12 (9), p.670-673.

2. Pockley, A. G., Shepherd J, Corton, J. M. Detection of Heat Shock Protein 70 (Hsp70) and Anti-Hsp70 Antibodies in the Serum of Normal Individuals. Immunol. Investig. 1998, 27, p.367-377.

3. Lee C. Y., D. A. Lappi, Wermuth C., J. Everse, Kaplan N. O. 8-(6-Aminohexyl)-Amino-Adenine Nucleotide Derivatives for Affinity Chromatography. Archives of Biochemistry and Biophysics. 1974, 163, p.561-569.

Claims

The method of determining the concentration of the primary heat shock protein 70 kDa (HSP70), which consists in the immobilization agent that interacts with HSP70 on the surface of the wells of 96-hole tablet for enzyme immunoassay, the washing of the wells with buffer, introduction into the wells of the studied sample or standard and incubation, washing of the wells, introduction into the wells of polyclonal rabbit antibodies to HSP70 and incubation, washing lunations, flushing holes, introduction into the wells of the substrate of the enzyme and Chromogen, incubation, spectrophotometric evaluation of developed color and correlate it with data calibration, characterized in that the immobilized agent that interacts with HSP70, use derived ATP covalently associated in a complex with HMDA with egg albumin, samples and standards add sodium salt of ATP to 10 mm, and also contribute to the clipboard magnesium ions to a final concentration of 10 mm in all phases.



 

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