RU2553370C1 - Method for determining c-reactive protein concentration by immune impedance analysis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: analysis uses a bipolar method for electrical impedancemetry to determine a module of the electrical impedance |Z| measured at a frequency of 20,000 Hz and low-intensity AC, a solution of monoclonal antibodies, analysed and reference samples. The concentration is calculated by solving a mathematical equation of relations of the analysed and known substance concentrations to the modules of the electrical impedance measured in a test well and a reference sample.

EFFECT: invention enables performing the method to avoid chemical agents and some laboratory equipment in addition to the monoclonal antibodies, and reducing the analysis time.

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Description

The invention relates to medicine, namely to laboratory diagnostics, and is intended to increase the efficiency of determining the concentration of C-reactive protein in blood serum and other biological fluids.

The closest analogue is the enzyme-linked immunosorbent assay, based on photometric measurement of the color intensity of a substrate solution associated with the immune complex of C-reactive protein and monoclonal antibodies, which changes the color of the solution in proportion to the amount of bound substance under the action of the myeloperoxidase enzyme (Clinical Laboratory Analytics Volume II. Private analytical technologies in the clinical laboratory / Under the editorship of V.V. Menshikov. - M.: Labinform-RAMLD, 1999. - P.201). An enzyme-linked immunosorbent assay for determining the concentration of C-reactive protein in serum is performed as follows. Serum samples are diluted with a buffer solution for diluting samples 100 times and mix thoroughly. 50 μl of diluted serum samples and control samples with known concentrations of C-reactive protein are added to the wells of an immunological plate with fixed monoclonal antibodies. 100 μl of monoclonal antibodies of a certain concentration conjugated to the horseradish myeloperoxidase enzyme are added to all wells, including control, containing known concentrations of C-reactive protein, and experimental. The plate is incubated for 30 minutes at room temperature. Perform the procedure of 3-fold washing with washing buffer solution, carefully remove moisture from the wells. 100 μl of a chromogenic substrate is added to each well, incubated for 30 minutes at room temperature, and then a stopping solution containing concentrated sulfuric acid is added. The color change of the solution in the wells is examined by photometry. Then, the concentration of C-reactive protein in each test sample of blood serum is calculated by comparing the obtained value with a calibration graph constructed on the basis of control samples with known concentrations of C-reactive protein.

The disadvantages of the prototype: the existing enzyme-linked immunosorbent assay is not effective enough, requires the use of monoclonal antibodies and additional expensive chemicals, as well as laboratory equipment, specially trained medical personnel (laboratory technician), has a duration of at least 2.5 hours, is intended for use in therapeutic and prophylactic institutions and not cost effective enough.

The invention is aimed at solving the problem - the simplification of the process.

, измеренного на частоте 20000 Гц переменного тока малой мощности, раствора моноклональных антител и исследуемого или контрольного образца, и расчет концентрации выполняют на основании отношений исследуемой и известной концентраций вещества к величинам модульного значения электрического импеданса, измеренным в опытной лунке и контрольной пробе.The problem is solved using the characteristics indicated in the claims common with the prototype, such as a method for determining the concentration of C-reactive protein in blood serum or other biological fluids in the wells of an immunological tablet, and distinctive, essential features, such as the bipolar method used for research electrical impedance measurements with determination of the modular value of electrical impedance $$\left|Z\right|$$

measured at a frequency of 20,000 Hz of alternating current of low power, a solution of monoclonal antibodies and a test or control sample, and the concentration calculation is performed on the basis of the ratios of the studied and known concentrations of the substance to the values of the modular value of electric impedance measured in the experimental well and control sample.

The technical result from the above set of essential features is to increase the efficiency of the method by eliminating the need to use chemicals and additional laboratory equipment to monoclonal antibodies, reducing the time to complete the study and reducing the cost of using the method.

. Одновременно, параллельно выполняется контрольный тест с внесением 50 мкл образца, содержащего известную концентрацию С-реактивного протеина в лунку, содержащую 250 мкл раствора моноклональных антител, аналогично опытной пробе. После этого рассчитывают концентрацию С-реактивного протеина в исследуемом образце (опытной пробе) на основании уравнения $$Соп/\left|Z\right|оп=Ск/\left|Z\right|к$$

и эквивалентного уравнения $$Соп=Ск\times \left|Z\right|оп/\left|Z\right|к$$

, где Соп - исследуемая концентрация С-реактивного протеина в опытной пробе, $$\left|Z\right|оп$$

- модульное значение электрического импеданса в опытной пробе, Ск - концентрация С-реактивного протеина в контрольной пробе с известной концентрацией, $$\left|Z\right|к$$

- модульное значение электрического импеданса в контрольной пробе с известной концентрацией С-реактивного протеина.The solution of this problem is achieved by measuring the electrical impedance of a solution of monoclonal antibodies, placed in the wells of a standard immunological tablet, with a volume of 300 μl, 250 μl each, followed by the addition of 50 μl of the test sample of blood serum, using the bipolar method using probing with low-power alternating current with a frequency of 20,000 Hz . To increase the accuracy of the measurement method, repeat at least 576,000 times for 3 seconds, and calculate the average value of the modular value of the electrical impedance $$\left|Z\right|$$

. At the same time, a control test is performed in parallel with the addition of 50 μl of a sample containing a known concentration of C-reactive protein in a well containing 250 μl of a solution of monoclonal antibodies, similar to the experimental sample. After that, the concentration of C-reactive protein in the test sample (test sample) is calculated based on the equation $$\mathrm{FROM}\mathrm{about}P/\left|Z\right|\mathrm{about}P=\mathrm{FROM}\mathrm{to}/\left|Z\right|\mathrm{to}$$

and the equivalent equation $$\mathrm{FROM}\mathrm{about}P=\mathrm{FROM}\mathrm{to}\times \left|Z\right|\mathrm{about}P/\left|Z\right|\mathrm{to}$$

where Sop is the test concentration of C-reactive protein in the test sample, $$\left|Z\right|\mathrm{about}P$$

- the modular value of the electrical impedance in the experimental sample, CK - the concentration of C-reactive protein in the control sample with a known concentration, $$\left|Z\right|\mathrm{to}$$

- the modular value of the electrical impedance in the control sample with a known concentration of C-reactive protein.

The method is as follows. Blood for examination is taken from the cubital vein or finger of the patient. Blood serum is isolated and 50 μl is added to a well of an immunological plate containing 250 μl of a solution of monoclonal antibodies using a pipette. The immunological plate is incubated for 30 minutes at room temperature. Electrodes of a hardware-software complex, for example, “BIA-lab Spiro” (RF Patent No. 2487662, 07/20/13, IPC A61B 5/08 publ. 07/20/13, or Medass ABC-01, or another (RF Patent “ A device for measuring the impedance of biological media "No. 2462185 from 07/19/2011. IPC A61B 5/08, publ. 09/27/12) connect to the electrodes placed in the wells of the immunological tablet, turn on the device in the mode of per second visualization of the results at a frequency of 20,000 Hz, then record the results of the study.The total recording time of at least 3 seconds, during which register . Less 576000 measurements result is written automatically in the email folder named by the patient names Total Time perform research in one well immunoassay tablet does not exceed 3-4 seconds and a standard immunoassay strip plate containing 8 holes, -. 12 seconds.

Diagnosis of the concentration of C-reactive protein in the test sample of blood serum can be carried out automatically using a computer program.

The error of the method for determining the concentration of C-reactive protein does not exceed 0.001 μg / ml, which is comparable with the results of enzyme immunoassay.

Case Studies

Example 1

Patient Ch., 53 years old, diagnosis: coronary heart disease, angina pectoris 2 f.k.

The concentration of C-reactive protein in blood serum, determined by enzyme immunoassay, is 2.8 μg / ml.

The method of immunoimpedance analysis determined the concentration of: C-reactive protein in the blood serum of 2.8 μg / ml.

The presented example demonstrates the high accuracy of the application of immunoimpedance analysis in the case of diagnosing the concentration of C-reactive protein in blood serum in clinical practice with a normal value of the determined parameter (less than 3 μg / ml).

Example 2

Patient S., 54 years old. Diagnosis: Cerebrovascular disease. The consequences of acute cerebrovascular accident in the pool of the left middle cerebral artery, transferred in 2010. Right-sided hemiparesis. Encephalopathy of vascular origin.

The concentration of C-reactive protein in blood serum, determined by enzyme immunoassay, is 6.1 μg / ml.

The method of immunoimpedance analysis determined the concentration of: C-reactive protein in the blood serum of 6.1 μg / ml.

The presented example demonstrates the high accuracy of the application of immunoimpedance analysis in the case of diagnosing the concentration of C-reactive protein in the patient's blood serum in case of exceeding the normal range.

Example 3

Patient P., 27 years old. Diagnosis: Community-acquired pneumonia, in the lower lobe on the left, not a severe course. ONE 1 tbsp.

The concentration of C-reactive protein in blood serum, determined by enzyme immunoassay, is 78.3 μg / ml. The method of immunoimpedance analysis determined the concentration of C-reactive protein 78.2 μg / ml.

The presented example demonstrates the high accuracy of the application of immunoimpedance analysis in the case of diagnosing the concentration of C-reactive protein in the blood serum of a patient with pneumonia and significantly exceeding the normal range of its concentration in the blood.

The proposed method differs from the known following features. The proposed method uses the determination of electrical impedance in the well of a standard immunological tablet with a volume of 300 μl containing 250 μl of a solution of monoclonal antibodies and 50 μl of the studied biological fluid (blood serum) or a control sample, which allows to determine the concentration of C-reactive protein based on the solution of the mathematical equation of relations the investigated and known concentrations of the substance to the values of the modular value of the electrical impedance measured in the experimental well and the control oh sample (hole).

From the description and practical application of the present invention, specialists will be obvious and other private forms of its implementation. This description and examples are considered as material illustrating the invention, the essence of which and the scope of patent claims are defined in the following claims, a combination of essential features and their equivalents.

Claims (1)

A method for determining the concentration of C-reactive protein in blood serum in the wells of an immunological tablet, characterized in that the bipolar method of electrical impedancemetry with determination of the modular value of the electrical impedance | Z | measured at a frequency of 20,000 Hz low-power alternating current, a solution of monoclonal antibodies, is studied and control samples, and the concentration calculation is performed based on the solution of the mathematical equation of the ratio of the investigated and known concentrations of the substance to elichinam modular electrical impedance values measured in the test well and the control sample.