RU2091794C1 - Immuno-conductometric system and sensor complex - Google Patents

Abstract

FIELD: facilities determining state and classification of biological material. SUBSTANCE: unit of sensors is filled with experimental and standard samples. Samples are connected to measuring generator by means of commutator, converted signal is compared with signal of reference generator. Information on frequency changes characterizes change of reactive component of solutions. There appears possibility of conducting both immune and biochemical diagnosis on basis of comparison of parameters of examined solution with standard one. EFFECT: expanded application field of proposed system and complex. 2 cl, 3 dwg, 2 tbl

Description

 The invention relates to means for determining the status and classification of biological material.

 Known systems containing conductometric meters associated with the indicator [1, 2] However, in this case they did not allow to obtain reliable information about the immune state of the body, since the accuracy of the systems was not high enough and it was not possible to quickly assess the state of the material.

Closest to the claimed one is a system that includes a sensor complex, a sensor unit connected to a switched input of a switch, the signal output of which is connected to a measuring generator, and the output of the latter is connected to an input of a control and signal processing device, the output of which is connected to an indicator board [3]
During the operation of such a system, the complex conductivity of biological material obtained as a result of specific preparation is recorded, but the system’s functioning process is also not efficient enough, without optimality and without the necessary standardization of material preparation methods, despite the fact that experimental and control samples are compared.

 Also known is a sensor complex, including a sensor insert made of plastic, on which there are protrusions sensitized by diagnostic antibodies, as well as a marker for diagnostic antibodies and the corresponding substrate [4] This complex is also closest in content to the declared one.

 However, in this case it is impossible to effectively and repeatedly use this insert with a high degree of reliability of the result.

 During the joint operation of the insert and the system, the complex conductivity of biological material obtained as a result of specific preparation is recorded, but the process of functioning of the system even in such a combination is not efficient enough, although conductometric measurements make it possible to register with high accuracy.

 The aim of this invention is to increase the selectivity and sensitivity in assessing the reactions of the antibody-atigen of the investigated solutions, increasing ease of use and improving operational parameters.

 Conductometry is based on measuring the electrical conductivity of liquid electrolytes, which is proportional to their concentration. Therefore, if you obtain conjugates of antibodies with substances that change the electrical conductivity of liquids, then in this way you can change the concentration of bodies.

 In the constant region of antibodies contains the carbon part. This allows, without affecting the binding, to attach colored, fluorescent markers, enzymes or polymer carriers to the carbon part after oxidation.

 Antibodies against cytomegalovirus and other antibodies were used as antibodies in the conjugate, and practical deoxyribonuclease 1, hydrolyzing double- or single-stranded DNA with the formation of a complex mixture of mono- to oligonucleotides with 5-phosphate rings, was used as their marker for the nucleic acid exchange marker.

In the presence of M ^{2+ ions,} DNKase 1 acts independently on each DNA chain, with the break points being randomly located.

High molecular weight deoxyribonucleic acid (DNK) (from salmon milk) with a relative molecular weight of at least 7 • 10 ^{6 was} used as a substrate for DNKase 1.

 Under the influence of an conjugate of antibodies, for example, cytomegalovirus, and DNase 1 on a substrate medium (DNA), a decrease in the viscosity of the medium and an increase in the number of infected groups by a factor of two should occur.

 The most common method for producing conjugates of antibodies with enzymes is the periodic oxidation of the carbohydrate component of immunoglobulin, followed by the interaction of the formed aldehyde groups with amino groups of proteins, leading to the formation of Schiff bases. The advantages of the method are the high reactivity of the aldehyde groups, the lack of formation of conjugates characterized by different molar ratios of the components as a result of crosslinking.

 A search conducted in patent and technical literature showed that the claimed populations are unknown, i.e. they comply with the patentability condition “novelty”.

 Since there is a need for such devices and they are made of known components, the claimed device meets the condition of "industrial applicability".

 And since as a result of using the claimed device the goal is realized, and such an implementation is not obvious to the average level of a specialist, the jammed device meets the condition of "industrial applicability".

 In FIG. 1 is a block diagram of a system; in FIG. 2 general view of the sensor unit; in FIG. 3 view of the holes of these sensors.

 The immune conductometric system includes a sensor unit 1, a starting device 2, moreover, the sensor unit 1 is connected to a switched input of the switch 3, the signal output of which is connected to the measuring generator 4, and the control input and output of the switch 3 are connected to the first output and the installation input of the starting device 2, a measuring generator 4, the input of which is connected to the signal output of the switch 3, a control and signal processing device, the input of which is connected to the output of the generator 4, contains a reference generator 5 and a program a removable divider 6, the outputs of which are connected to the comparison element 7, the I8 element, the converter 9, moreover, the output of the element 7 is connected to the first input of the I8 element, to the second input of which the second output of the starting device 2 is connected, and the output of the I8 element is connected to the input of the converter 9 , the output of which is the output of the control and signal processing device and is connected to the input of the indicator board.

 The sensor unit 1 is a cage 11 with the corresponding wells-containers 12 and 13 for the control and prototypes, respectively, the contact zones 14 and 15 in these containers by means of contact tracks 16 are connected to the collector zones 17 and 18. The inner surfaces 19 of the holes 12 and 13 are made ribbed to increase the contact area for the sensitized reagent.

 For operation, the sensor block is equipped with a sensor complex made in the form of a sensor insert in the form of a central rod, in the lower part of which ribs (not shown) are evenly distributed, the surface of the ribs being sensitized with diagnostic antibodies, pancreatic deoxyribonuclease 1 was used as a marker of these antibodies, and high molecular weight deoxyribonucleic acid with a relative molecular weight of more than seven was used as the corresponding substrate in the calibration solution millions.

 The immune-conductometric system and the sensor complex work as follows.

 Before starting, a selection of specific sensory objects for the proposed diagnosis is made. The necessary processing is preliminarily performed, the coating of the insert and / or surface of 19 wells 12 and 13 is sensitized with test antibodies, or with necessary reagents. The test samples are prepared, and dilution can be fractions of a millionth part. Sampling is carried out, for example, in well 13, a control liquid is placed in well 12. Turn on the starting device 2. The enabling signals are fed to switch 3 and circuit I8. The program of work of the switch 3 includes the possibility of temporary switching from the collector zones 17 to the collector zones 18 with the appropriate indication and return back. The switching time is about 15 seconds. The first 15 seconds of the start of operation after the starting device 2, the collector zones 17 of the control sensor are connected through a switch 3 to the measuring generator 4 and, taking into account the specific solution, the frequency of the generator 4 is set in accordance with the magnitude of the reactive component of this control solution. The measuring frequency generated using the calibration solution selected specifically for this diagnosis through the divider 6 is fed to element 7, for example, a programmable divider, the second input of which is supplied with frequency from the reference generator 5. Comparison element 7, for example, a programmable divider or counter, and the reference generator 5 is made similarly to the generator 4. The signal from the element 7 enters through the I8 element to the converter 9 and then to the indicator board 10, which allows to obtain information about the quality of the analyzed sample and the amount of the analyzed component. The calibration test of the calibration sensor determines for the operator, either in the case of biochemical analysis or for the immunological evaluation of the antibody-antigen reaction, the characteristic of the negative antibody-antigen reaction. After 15 seconds of evaluating the calibration solution, switch 3 connects the "experience" sensor and the measurement process is repeated with the only difference that the indicator board displays information on the amount of analyte for analysis and the quality of the antibody-antigen reaction for the antibody-atigen immunological reaction for the immunological reaction, characteristic of the test sample. After the next 15 seconds of evaluating the experience, the switch 3 generates a signal to reset the starting device 2. The analysis process in comparison with the calibration sample is completed. The operator receives information in one case - for an immunological assessment, a positive or negative reaction in comparison with a control negative reaction, or the amount of the desired component in the case of biochemical analysis during preliminary calibration of the analyzer with a solution containing the desired component. For scoreboard 10, correction factors are introduced in each case of analyzing a specific substance, for example, for assessing glucose in blood or urine, for assessing bilirubin in blood and urine, for assessing nitrates, etc.

 Experimental part.

Example 1. To a solution of 24 mg of antibodies to cytomegalovirus (SMU) in 4 ml of water (6 mg / ml) was added a solution of 32 mg of Na ₂ SO ₄ in 4 ml of water (8 mg / ml) and kept for 30 min at room temperature. Then, 0.8 ml of 10 aqueous ethylene glycol was added to decompose the sodium periodate. Excess low molecular weight components were removed by dialysis at 4 ^{° C.} in water overnight. The SMU-DNKase 1 antibody conjugate was prepared by mixing 8 ml of a solution of oxidized antibodies with 8 ml (5 mg / ml) of pancreatic DNKase 1 in 0.1 M bicarbonate buffer (pH 9.5). The reaction mixture was incubated overnight at 4 ^{° C.} , then dialysis was performed against water for one day. The resulting conjugate was treated with a NaBH ₄ solution (4 mg / ml) for 1 h in the cold. Then dialyzed against distilled water at 4 ^o C. Theory-SMU 3 mg / ml

Thus, the main scheme for evaluating diagnostic antigens is as follows:
Substrate
At + Ar + At (Farm. + Label)
Product.

 Immune conductometric sensor works as follows.

Before starting a diagnostic assessment of the antibody-antigen immunological reaction using prepared sensory inserts sensitized with the appropriate diagnostic antibodies (for assessing viral, bacterial, parasitic or somatic diseases), either the patient’s blood serum or saliva, urine, lymph, etc. are obtained in a known manner. d. in an amount of 50 to 100 μl and dilute this biological fluid to 10 ^-6 - 10 ^{-9 with} either physiological saline or immunological buffer solution.

In the process of evaluating a specific immune response, an immune conductometric sensor is divided into the following steps:
1. Antigen recognition, i.e. sensitized on a polystyrene insert test antibodies are placed in the test solution (blood serum, urine, saliva), previously diluted 10 ^-6 times. The solution is taken in a volume of 200 μl. The time spent by the sensor insert in the solution is 15 minutes

 2. Washing the sensor insert in distilled water for 1 min.

3. The step of linking the desired antigen with test labeled antibodies. After washing in distilled water, the sensor insert is placed in the well of the plate with a solution of labeled test antibodies, i.e. sensory insertion (in the presence of the desired antigen) is as follows:
sensitized test antibodies + desired antigen + labeled test antibodies (At + Ar + At.met).

 The residence time of the sensor insert in solution with labeled test antibodies is 15 minutes. Then the sensor insert is washed in distilled water for 1 min.

 4. The step of processing the label of test antibodies in the calibration solution.

 Touch insert with "At + Ar + At.met." placed in a well with a calibration solution for 3 to 5 minutes In this solution, the chemical reaction of the label with the components of the calibration solution occurs, which leads to larger or smaller (depending on the number of associated labeled test antibodies with the desired antigen) changes in the electrical properties of the calibration solution.

 5. The stage of assessment of changes in the electrical properties of the calibration solution.

 The sensor insert is removed from the calibration solution and placed in a weak solution of acetic acid for 1 min, then washed in distilled water in order to remove the "Ar + At. Met." for further use of this sensory insert in the diagnosis of the desired antigen.

 A conductometric sensor is placed in a free well (after sensory insertion) and the results obtained are recorded. Depending on the magnitude of the changes in the electrical properties of the calibration solution, referred to the "At + At + At.met." Bond introduced by the sensor insert into the calibration solution, three negative points are recorded (in the absence of the "Ar + At.met. "), Or from one to three" pluses "depending on the number of ligaments" At + Ar + At. Met. ".

 The total preparatory time for diluting the test solutions varies from 1.5 to 2.5 hours, depending on the number of samples (up to 50 samples) and the preparation of the laboratory assistant. Analysis time (obtaining the final result) 40 min.

 Thus, the total evaluation time of 50 samples varies within 2 hours.

 Examples of ongoing assessment of herpes virus and cytomegalovirus on the basis of the Institute of organ and tissue transplantation (Table 1).

 Herpes virus was evaluated on the basis of the 1st ICD in comparison with the assessment of molecular hybridization of the same donor serum samples. Assessment using molecular hybridization (probes) was carried out on the basis of the Gamalei Institute (Table 2).

The positive qualities of the immune conductometric sensor, in comparison with the analogue and prototypes, include the following:
reduction of the effect of cross-reactions of the antibody-antigen due to the large dilution (10 ^-6 -10 ^-9 ) and the use of sensory insertion, and, as a result, increased immune selectivity;
an increase in sensitivity (from 100 to units of pictograms of the diagnosed antigen) due to the use of large dilutions and conductometric evaluation of the calibration solution (this is clearly seen in comparison with the data of molecular hybridization in the assessment of herpes);
the use of a small amount of expensive test material (diagnostic antibodies) in sensitization of sensors, the repeated use of sensor inserts;
short time to obtain the final result (2 h of 50 samples) With an increase in the number of samples, the increase time increases only due to the experience and professionalism of the laboratory assistant);
the availability and ease of use of the immune conductometric sensor;
manufacturability, low cost of receiving sensor inserts.

Information sources:
1. US patent N 5082550, CL. G 01 N 27/00, 1992

 2. RF patent N 2027174, cl. G 01 N 27/02, 1995

 3. Application of France N 2598227, CL G 01 N 33/53, 1987

 4. US patent N 4977078, cl. G 01 N 33/53, 1990

Claims (2)

 1. An immunoconductometric system including a sensor complex, a sensor unit connected to a switched input of a switch, the signal output of which is connected to a measuring generator, the output of the latter being connected to an input of a control and signal processing device, the output of which is connected to an indicator board, characterized in that the device control and signal processing is made in the form of a starting device, AND circuit, converter, as well as a programmable divider, the input of which is the input of the control device signal and reference generator, the outputs of the last two connected to the corresponding inputs of the comparison element, the output of which and the first output of the starting device are connected to the inputs of the circuit AND, the output of the latter is connected to the input of the converter, the output of which is the output of the control and signal processing device, in addition, the second output of the starting device is connected to the control input of the switch, the control output of which is connected to the installation input of the starting device.  2. A sensor complex, comprising a sensor insert made of plastic and containing protrusions, the surface of which is sensitized with diagnostic antibodies, a marker for diagnostic antibodies and an appropriate substrate, characterized in that the insert is made in the form of a central rod, in the lower part of which there are ribs and adjacent ones established with constant angular displacement relative to each other, pancreatic deoxyribonuclease 1 was used as a marker for diagnostic antibodies, and as a corresponding In the calibration solution, a high molecular weight deoxyribonucleic acid with a relative molecular weight of more than seven million was used.

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