RU2176794C2 - Method for setting infection diagnosis - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves subjecting patient blood to diagnostic examination in determining microbial antigen quantity bound by blood erythrocytes in indirect way by applying polarography method, by making comparison to control study results and taking into account the degree of reduction in copper ion wave bound to this antigen. EFFECT: accelerated and simplified diagnosis method. 7 dwg

Description

 The invention relates to medicine and can be used to diagnose acute viral and bacterial infections.

 There is a method of immunodiagnosis of infections by determining the phagocytic activity of leukocytes of two samples of a patient’s blood when it is incubated with a specific object of phagocytosis with the addition of a soluble specific microbial substance to one of the samples, then the index of change in the phagocytosis of a specific object is calculated by the levels of phagocytosis in the sample with additional exposure to a specific microbial substance in relation to the indices of the sample without additional microbial exposure and with an index value of less than 0.75, the diagnosis infection infection (A.S. USSR N 1789927, MKI G 01 N 33/53). The method of immunodiagnosis of infections / V.N. Kaplin, E.I. Samodelkin, V.F. Kolomoytsev (USSR). - N 4866935/14; Stated September 11, 90; Publ. 01/23/93. Bull. N 3 // Discoveries. Inventions - 1993.- N 3.- C.12.

 However, the visual count of phagocytosis objects is of a certain subjective nature and requires a certain amount of time. In this regard, hardware methods for determining the degree of antigen binding are of particular interest.

 The invention is aimed at accelerating, simplifying the method with high sensitivity and specificity.

 The essence of the invention: this task is achieved by the fact that a diagnostic study is carried out with the patient’s blood, using the polarographic method to determine the amount of microbial antigen bound by the patient’s blood cells before and after additional exposure of the blood cells to the same antigen by changing the wavelength of copper ions associated with this antigen. The study is not a plasma, but the sensitivity of blood cells to various microbial antigens provides the diagnosis of infectious diseases even in the earliest possible time from the moment of infection.

 It is known that polarographically inactive substances can be detected if an active group is introduced into the test compound or determined indirectly by a decrease in the wave of the reducing substance, which gives a precipitate or complex with the test compound.

 A polarographic study allows you to quantitatively (after drawing up the calibration curve) to determine the degree of binding of antigen to blood cells by decreasing the micro amount of the number of copper ions. In turn, a comparison of the polarograms of healthy control donors and sick people allows us to make a diagnosis.

As a background solution using 30% CaCl ₂ in a volume of 4.8 ml. 10 gamma divalent copper (0.1 ml of a standard solution with a concentration of 100 gamma copper in 1 ml) is added to the electrolyzer and, after mixing, the polarogram is recorded three times.

 After that, 0.1 ml of the test solution is added directly to the electrolyzer, mixed and polarogram recorded, measuring the half-wave in mm. Based on a pre-made calibration, the amount of antigen remaining in the solution after interaction with red blood cells is calculated and the disease is diagnosed.

The method is illustrated by drawings of polarograms, where in FIG. 1 a shows the polarogram of a background solution of 30% CaCl ₂ ;
- FIG. 1 b - combined polarogram of the background solution and 10 gamma Cu ⁺⁺ solution;
- FIG. 2 - polarogram of the background solution and 10 gamma Cu ⁺⁺ solution;
- FIG. 3 - polarogram after introducing into the cell 0.1 ml of the studied antigen;
- FIG. 4 polarograms after introducing into the electrolyzer 0.1 ml of the test antigen in contact with the blood of healthy individuals without additional exposure to the antigen;
- FIG. 5 - polarogram after introducing into the electrolyzer 0.1 ml of the test antigen in contact with the blood of healthy individuals with additional exposure to the antigen;
- FIG. 6 - polarogram after introducing into the electrolyzer 0.1 ml of the test antigen in contact with the blood of sick people without additional exposure to the antigen;
- FIG. 7 - polarogram after introducing into the electrolyzer 0.1 ml of the test antigen with the blood of patients with additional exposure to the antigen.

 The method is as follows.

For the study, 0.1 ml of blood is taken, mixed with a preservative (0.1 ml of Glyugitsir), centrifuged for 5-10 minutes at 500 rpm, the plasma with anticoagulant is removed, blood cells are washed three times from plasma residues 0.9 % solution of sodium chloride, dilute blood cells in a 0.9% solution of sodium chloride to 5-10%, in 2 samples of 0.1 ml of this suspension add 0.1 ml of the test antigen (influenza, Koksaki B enterovirus , Shigella Flexner, Sonne, Newcastle or others), mix and incubate the resulting mixture for 3-5 minutes in an thermostat at 37-37.5 ^o C, then one sample trifugate, and then remove the polarogram 10 gamma CuCl ⁺⁺ in the background solution of 30% CaCl ₂ , adding 0.1 ml of the supernatant, again remove the polarogram, taking it as ASAE without additional exposure to the microbial antigen. In parallel, 0.1 ml of the same antigen is re-added to the patient’s second blood sample, mixed and re-incubated at 37-37.5 ^o C, centrifuged, then the polarogram is removed as described above and the polarogram values are compared before and after the additional exposure of the antigen to blood cells . In healthy individuals, an additional antigen exposure causes an increase in ASAE, whereas in patients, the antigen-binding activity of red blood cells is inhibited after an additional exposure to the same antigen.

The cell of the electrograph of the polarograph is filled with a background solution of 4.8 ml of 30% CaCl ₂ . When voltage is applied to the electrodes of the electrolyzer in the background solution of the polarograph cell, an electric current appears, which is recorded by the polarograph recorder in the form of a standard curve (Fig. 1 a), and when the gamma Cu ⁺⁺ is introduced into the electrolyzer 10, a deviation in the form of a half wave is observed (Fig. 1 b )

The magnitude of the current depends on the amount of electrically conductive ions in the solution of the cell of the polarograph electrolyzer, which in our study are the CaCl ₂ ions _{of the} background solution and the added micro amount of divalent copper ions - 10 gamma Cu ⁺⁺ (Fig. 2).

 The polarograph recorder graphically displays the magnitude of the current between the electrodes of the polarograph cell on a paper tape. The relationship between the magnitude of the electric current and the number of copper ions added to the cell is determined by the degree of deviation of the pen of the recorder on paper. On this basis, a calibration curve is made by measuring the deviation of the pen of the polarograph recorder after introducing 0.1 ml of the studied antigen into the electrolyzer, which, by binding copper ions, reduces the wavelength (Fig. 3). The value by which the wavelength decreased is taken as 100% of the calculated value of the degree of antigen binding.

After drawing up the calibration curve, the ability of the blood cells of healthy individuals to bind the studied antigen on their surface is examined. For this, blood cells are diluted in a 0.9% sodium chloride solution, the test antigen is added, and after incubation in a thermostat of blood cells with antigen, this mixture is centrifuged. The supernatant is sucked off with a micropipette and introduced into the cell of a mercury-droplet polarograph, into which a background solution of 30% CaCl ₂ with 10 gamma Cu ⁺⁺ is preliminarily added. When voltage is applied to the electrodes of the electrolyzer, an electric current appears in the solution, which causes the pen of the recorder to deviate from the standard curve of the background solution. Research parameters are always constant. In this case, the current value depends on the amount of electrically conductive ions in the solution of the cell of the electrograph of the polarograph, which are Cu ⁺⁺ ions in our study. To determine the degree of binding of the studied antigen by the blood cells of healthy individuals, the electric current is divided by the degree of deviation of the recorder’s pen on paper in millimeters by the value in mm obtained when determining the standard, and the percentage of binding of the amount of antigen in this study is determined before and after additional exposure to the antigen. In healthy individuals, the degree of erythrocyte binding of the antigen after additional exposure to the same antigen increases, while in the patient after additional exposure to the antigen, its binding decreases due to inhibition.

Calculation example:
FIG. 2) the magnitude of the polarogram wave of 10 gamma copper in 30% CaCl ₂ = 160 mm;
FIG. 3) this value decreased after adding antigen to 100 mm, so when adding 0.1 ml of the studied antigen, the wave decreased by 60 mm, which is taken as 100% of the variable when adding antigen;
FIG. 4) when recording a polarogram of a healthy person after initial contact with an antigen, the wave size was 110 mm, respectively 110 - 100 = 10 mm: 60 mm = antigen binding of 16.6%;
FIG. 5) when recording a polarogram of a healthy person after repeated contact with an antigen, the wave size was 123 mm, respectively 123-100 = 23 mm: 60 mm = 38.3% (ASAE level increased even more - a healthy donor);
FIG. 6) when recording the polarogram of a sick person after initial contact with an antigen, the wave size was 130 mm, respectively 130-100 = 30: 60 (ASAE level immediately reached 50% of the antigen dose - a sick person);
FIG. 7) when recording the polarogram of a sick person after additional exposure to an antigen, the wave size was 110 mm, respectively 110-100 = 10 mm: 60 mm = 16.6% (ASAE level decreased - a sick person).

 Examples of specific performance.

Example 1. Patient I-nov (43 years old) was admitted to the infectious diseases department of the OKB with complaints of fever, headache, and abdominal pain. Sick 3rd day. Preliminary diagnosis: "Enterovirus infection." Take 0.1 ml of the patient’s blood, mix with a preservative (0.1 ml of Glyugitsir), centrifuge for 5-10 minutes at 500 rpm, remove the plasma with anticoagulant, wash blood cells from plasma residues 0.9% three times solution of sodium chloride, blood cells are diluted in a 0.9% sodium chloride solution to 5-10%, 2 samples of 0.1 ml of this suspension are mixed with 0.1 ml of the studied antigen (Coxsackie enterovirus B), the incubated the mixture for 3-5 minutes in a thermostat at 37-37.5 ^o C, one sample is centrifuged, after which the polarogram 10 gamma Cu ⁺⁺ is removed in the background solution of 30% -N Oh CaCl ₂ (wavelength 160 mm) and, adding 0.1 ml of the supernatant of the first sample, again remove the polarogram (wavelength 135 mm). The magnitude of the wave with 0.1 ml of the studied antigen (Koksaki B enterovirus) is 105 mm, thus 135-105 = 30 mm: 55 mm x 100% = 54.5% is connected with the patient’s red blood cells.

In parallel, 0.1 ml of the same antigen is re-added to the second blood sample of the patient, mixed and re-incubated at 37-37.5 ^o C, centrifuged, and then the polarogram is removed as described above. The magnitude of the wave is 105 mm, thus the patient erythrocytes are connected 105-105 = 0 mm: 55 mm x 100% = 0.0% (inhibition of ASAE after additional exposure to the same antigen is characteristic of a sick person.

 Conclusion: the patient has an enterovirus infection (the diagnosis was later confirmed serologically and clinically).

Example 2. Donor S-kin (52 years old). Take 0.1 ml of the patient’s blood, mix with a preservative (0.1 ml of Glyugitsir), centrifuge for 5-10 minutes at 500 rpm, remove the plasma with anticoagulant, wash blood cells from plasma residues 0.9% three times -sodium chloride solution, dilute blood cells in a 0.9% sodium chloride solution to 5-10%, mix 2 samples of 0.1 ml of this suspension with 0.1 ml of the studied antigen (Coxsackie enterovirus B), incubate the obtained the mixture for 3-5 minutes in an thermostat at 37-37.5 ^o C, one sample is centrifuged, after which the polarogram 10 gamma Cu ⁺⁺ is removed in a 30% -n background solution Oh CaCl ₂ (wavelength 160 mm) and, adding 0.1 ml of the supernatant of the first sample, again remove the polarogram (wavelength 115 mm). The magnitude of the wave with 0.1 ml of the studied antigen (Koksaki B enterovirus) is 105 mm, thus 115-105 = 10 mm are connected with red blood cells: 55 mm x 100% = 18.2% (the level of binding of a healthy person).

In parallel, 0.1 ml of the same antigen is re-added to the second blood sample, mixed and re-incubated at 37-37.5 ^o C, centrifuged, and then the polarogram is removed as described above. The magnitude of the wave is 125 mm, so the patient’s erythrocytes are associated with 123 - 105 = 20 mm: 55 mm x 100% = 36.4% (the ASAE level increased after additional exposure to the antigen - this is a healthy person).

 Conclusion: the examined enterovirus infection does not.

 Conclusion: given that the main methods for diagnosing infections are clinical and serological methods, the results of which are only retrospective (determining the degree of increase in antibody titers in "paired" sera is done 10-15 days after the onset of the disease), or the isolation and identification of the pathogen in the material from a sick person (which is very laborious and takes a long period of time), and the claimed method allows you to diagnose the disease within 25-30 minutes from the start of the examination of the patient and It can be used for differential diagnosis of diseases similar to it according to clinical signs.

 The proposed method is accurate, with repeated polarographic studies, the results were almost the same.

 The proposed method is objective, because the conclusion about the presence of the disease is made on the basis of the magnitude of the polarographic wave of the antigen of the supernatant, which is carried out by simple measurement using a millimeter ruler, excluding any subjectivity in evaluating the results.

 The proposed method significantly reduces the complexity and increases labor productivity in comparison with analogues.

Claims (1)

A method for the immunodiagnostics of infections, including taking a blood sample, obtaining red blood cells, followed by adding to them a 0.9% solution of sodium chloride, and then a solution of the test antigen, incubating the mixture and determining the diagnosis of a significant indicator, characterized in that for two samples of a mixture of red blood cells and sodium chloride is added the test antigen solution at a volume ratio of 1: 1, the mixture obtained is stirred and incubated for 3-5 min at 37-37,5 ^o C, then the first sample is centrifuged, removed polyarogram at 10 ug Cu ⁺⁺ ions in 30% strength solution of ₂ SaS1 after adding supernatant and taking as the standard, and the second sample introduced further antigen mixture was re-stirred and incubated for 3-5 min at 37-37,5 ^o C, then centrifuged, removed polarogram 10 g Cu ⁺⁺ ions in 30% strength solution of ₂ SaS1 after adding the supernatant, and compared with the reference polarograms and decreasing the degree of deviation of the pen recorder in polarograms after further addition of the antigen in comparison with the deflection of the recorder pen on the reference polarogram diagnose the disease.

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