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 , 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 , 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 , 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.
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.
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.
man, is able to induce the formation of neutralizing antibodies to human tnfdna, its coding, vector (options), a method of obtaining a vaccine tnf(options), method of testing for the presence of tnfthe way to test body fluids of a person, the method of diagnosis, method of treatment and prophylaxis medication for the treatment of" target="_blank">
FIELD: medicine, ophthalmology.
SUBSTANCE: in lacrimal liquid one should detect the content of interleukin 8 (IL-8) and that of interleukin 1 beta (IL-1β) to calculate prognostic coefficient (PC) due to dividing the first value by the second one by the following formula: At PC value being below 10.0 one should predict favorable disease flow, and at PC value being above 10.0 - unfavorable flow.
EFFECT: higher accuracy of prediction.
FIELD: medicine, medicinal microbiology.
SUBSTANCE: method involves growing microorganism culture to be studied in solid nutrient medium followed by preparing microbial suspension and its incubation in the presence of lactoferrin. Control sample is prepared in parallel series. Control and experimental samples are incubated, supernatant is removed from bacterial cells and lactoferrin concentration is determined in supernatant of experimental and control sample by immunoenzyme analysis. Then anti-lactoferrin activity is calculated by difference of concentrations of residual lactoferrin in experimental and control samples. This method provides enhancing the sensitivity and precision in carrying out the quantitative evaluation of anti-lactoferrin activity in broad spectrum of microorganisms that is urgent in diagnosis and prognosis of diseases with bacterial etiology. Invention can be used in determination of persistent indices of microorganisms for assay of their etiological significance in pathological processes.
EFFECT: improved assay method.
3 tbl, 3 ex
FIELD: medicine, biology.
SUBSTANCE: invention relates to nutrient medium used for accumulation of cells for the following cytological and/or immunocytochemical analysis carrying out. Invention relates to medium containing salts NaCl, KCl, anhydrous CaCl2, MgSO4 x 6 H2O, MgCl2 x 6 H2O, Na2HPO4 x 2 H2O, KHPO4, NaHCO3, and also glucose and Henx's solution, 10% albumin solution and polyglucin taken in the ratio 1:1:1. Invention provides enhancing the preservation of cells.
EFFECT: improved an valuable properties of nutrient medium.
FIELD: medicine, cardiology.
SUBSTANCE: in peripheral blood one should detect the level of CD95(+) and CD16(+) neutrophilic granulocytes and at combination of increased level of CD95(+) neutrophilic granulocytes by 4 times and more and CD16(+) neutrophilic granulocytes by 0.6 times against the norm with ECG signs of myocardial infarction one should predict lethal result of large-focal myocardial infarction.
EFFECT: higher accuracy of prediction.
FIELD: medicine, parasitology.
SUBSTANCE: one should carry out immunoenzymatic assay to detect diagnostic optic density and that of labeled immune complex in a plot's hole with tested serum measured in conventional units at wave length being 492 nm. One should calculate coefficient of antibodies concentration measured in conventional units by the following formula: CAC = (Odtsh - Odd) x 100, where CAC - coefficient of antibodies concentration, Odtsh - optic density of the hole with tested serum, Odd - diagnostic value of optic density, 100 - coefficient of serumal dilution. By CAC value one should detect the titer of antibodies to Lamblia intestinalis antigens to interpret results of the trial. The method enables to study the dynamics of disease flow.
EFFECT: higher efficiency and accuracy of diagnostics.
1 ex, 1 tbl
SUBSTANCE: the present innovation deals with studying and treating diseases of inflammatory, autoimmune and degenerative genesis. One should perform sampling of heparinized blood followed by its sedimentation to obtain blood plasma with leukocytes and centrifuging to isolate the latter which are washed against erythrocytic and serumal admixtures, and, also, it deals with calculating the number of cells in samples out of leukocytic suspension after incubation (B) for 1.5 h at 37 C in holes of plastic microplotting board, out of leukocytic suspension one should additionally prepare two samples, one should be applied to calculate total number of leukocytes before incubation (A), the second sample undergoes incubation at the same mode at addition of autoserum to calculate the number of cells remained after incubation (C). One should state upon adhesive properties of leukocytes by the index of spontaneous adhesion (D), where D=(A-B)/B.100%, and effect for enhanced cellular adhesion under the impact of autoserum should be detected by the value of K=(B-C)/C.100% at K ≥ 30%, where B - C - the number of cells undergone additional adhesion after addition of autoserum. The present innovation widens functional possibilities of the suggested method due to obtaining additional values depicting adhesive properties of blood leukocytes.
EFFECT: higher accuracy of detection.
FIELD: medicine, immunology.
SUBSTANCE: one should carry out reaction of blast-transformation, detect proliferation of T-lymphocytes activated with antibodies to CD3 in the presence of interleukin-7 (ACT IL-7) and in the presence of interleukin-7 and dexametazone (ACT IL-7 D), calculate the index for dexametazone action as the ratio of ACT IL-7 to ACT IL-7 D, moreover, the value of dexametazone action index being above 1.2 indicates increased production of cytokins that suppress T-lymphocytes in neonatals. The method enables to detect functional defect of immune system that characterizes neonatal period.
EFFECT: higher efficiency of detection.
SUBSTANCE: method involves measuring forced exhalation volume per 1 s (FEV1) in l, full right ventricle evacuation time (RVE) in ms and angiotensin II value (AII) in ng/l. Discriminant relationship is built as D=0.504·RVE+3.038·FEV1 - 2.0·AII. D being less than 83.88, pulmonary hypertension occurrence is predicted within 1 year. D being equal to or greater than 83.88, no pulmonary hypertension is predicted to occur.
EFFECT: enhanced accuracy of prediction.
FIELD: medicine, medicinal immunology.
SUBSTANCE: method involves determination of heterophilic antibodies in human serum blood by the Paul-Bunnel's method relatively the level of circulating immune complexes, complement-activating properties of heterophilic antibodies by incubation of standardized ram erythrocytes with 0.8% serum for 30 ± 5 min and the following measurement of the erythrocytes lysis degree. The measurement of the effector function coefficient of heterophilic antibodies is carried out by the complement system Keff.f.h.a.-c.s. by the formula: Keff.f.h.a.-c.s. = Y/Tg.a. wherein Y means a lysis degree, %; Tg.a. means a reverse titer of heterophilic antibodies to ram erythrocytes. The damage assay is carried out by comparison of the immune status with the relative level of circulating immune complexes in serum. Method provides detection of preclinic from of immunodeficiency and autoimmune diseases that opens the possibility for their prophylaxis at most early stages of development. Invention can be used for assay of damage in the immune status in human serum blood.
EFFECT: improved method for assay.
5 tbl, 1 ex
SUBSTANCE: method involves concurrently examining anti-inflammatory IL-4 level in blood serum and lacrimal fluid. The value being within the limits of 60-70 pg/l in blood serum and 5-15 pg/l in lacrimal fluid, disease prognosis is considered to be unfavorable. The IL-4 concentration being within the limits of 90-100 pg/l in blood serum and 20-30 pg/l in lacrimal fluid, disease prognosis is considered to be favorable.
EFFECT: high accuracy of diagnosis.