RU2495433C2 - Kit of reagents for immune-enzyme assay of endogenous bioregulator antibodies in blood serum for detecting addiction disorders - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is presented is a kit of reagents for immune-enzyme assay of the endogenous bioregulator antibodies in blood serum for detecting addiction disorders by two-site enzyme immunoassay in human blood serum in vitro. The kit comprises a 96-well demountable stripped plate with its surface sequentially coated by all conjugates: βendorphins, serotonin, dopamine, histamine, orphanin and angiotensin, as well as containers containing positive and negative reference samples, horseradish peroxidase-labeled anti-species anti-human antibodies, phosphate-buffered saline containing Tween 20, a substrate buffer solution, a tetramethyl benzidine substrate solution, and a stop solution.

EFFECT: more accurate detection of the addiction diseases, including alcoholism, drug addiction, chemical abuse, food addiction and gambling by using the kit of six labels.

3 tbl, 9 ex

Description

The invention relates to medicine and can be used to identify individuals with various addiction diseases, including: toxicochemical addiction: alcoholism, drug addiction, substance abuse; food addiction, gambling (gambling).

Currently, there has been an increase in the number of people with various forms of addiction diseases, with the manifestation of a clinic of pathological attraction to the use of drugs, drugs, alcohol, gambling, food, etc. These people need timely diagnosis and the provision of treatment and rehabilitation assistance.

Diseases of dependence are classified as neuroimmunochemical pathologies. Their development is accompanied by a violation of the interaction of the central nervous and immune systems.

Each psychoactive drug has its own pharmacological spectrum of action. However, all substances that can cause addiction syndrome have a common link of pharmacological action - this is a characteristic effect on catecholamine (CA) neuromediation in the limbic structures of the brain, in particular in the reinforcement system.

Exposure to psychoactive substances (surfactants) leads to an intensive release of neurotransmitters from the CA group from these brain regions, primarily dopamine (DA), and, consequently, to a much stronger excitation of the reinforcement system. Such arousal is often accompanied by positively colored emotional experiences. Free spacecraft are exposed to metabolic enzymes and are rapidly destroyed. Repeated surfactant methods lead to depletion of neurotransmitter reserves, which is manifested by insufficiently expressed excitation of the reinforcement system upon receipt of a “normal” impulse. Psychophysically in humans, this is expressed by a drop in mood, a feeling of lethargy, weakness, feelings of boredom, emotional discomfort, and depressive symptoms. Taking surfactants against this background again causes an additional release of neurotransmitters from the depot, which temporarily compensates for their deficiency in the synaptic cleft and normalizes the activity of the limbic structures of the brain. This process is accompanied by a subjective sensation of improvement, emotional and mental arousal, etc. However, free spacecraft are rapidly destroyed, which leads to a further drop in their content, worsening of the psychoemotional state and, accordingly, to the desire to reuse surfactants.

Early diagnosis of such conditions is very important, because besides all these diseases have a social aspect.

A known method for determining a predisposition to the formation of opiate dependence, in which the in vitro concentration of cyclic adenosine monophosphate (cAMP) is determined under a test load of morphine in combination with naloxone in native and in native pre-morphinated mononuclear peripheral blood cells, the predisposition index is calculated by dividing the concentration value of cAMP in previously morphinated to its value in native mononuclear cells and with an index value of more than 1.3 elyayut high predisposition to the formation of opioid dependence. (RU, C1, 2177157, MKI 7 G01N 33/68, publ. 2001.12.20)

This method has high accuracy, but is complex and determines only opiate dependence.

The prior art method for predicting the risk of additive, compulsive and impulsive disorders in the form of alcoholism, obesity and smoking by molecular genetic studies of the DRD2 gene (Pat. US 5550021, 08.27.1996).

However, conducting a genetic research is a long, costly process that requires expensive equipment and highly qualified specialists.

A known method for determining the predisposition to diseases of dependence on the presence of natural antibodies to endogenous bioregulators in the blood of healthy and sick people suffering from various types of addiction, gambling addiction, alcoholism, obesity. It has been established (RF patent 2369872, IPC G01N 33/53 (2006.01), publ. 10.10.2009) that patients with addiction diseases have a significantly increased or decreased level of antibodies. During treatment, it can change and correlate with a person’s state of health. Based on this fact, a deviation from the norm in the antibody content in the group of healthy individuals can be regarded as a biological risk factor, which in a certain situation leads to addiction disease.

These factors must be identified in advance in order to plan an individual health care program for these people, recommend a gentle professional orientation, etc.

In the known method, the test sample is incubated with a hapten conjugate: a macromolecular carrier immobilized on a solid phase, then the formed immune complex is incubated with a conjugate of antibodies against human immunoglobulins of class M and G labeled with an enzyme, and the conjugate in which the hapten refers to a group of opioid peptides, or biogenic amines or peptides of the renin-angiotensin system, and the macromolecular carrier has a protein or polymer nature

The panel of antigens included endogenous bioregulators belonging to the following classes - opioid peptides: (β-endorphin, dermorphin, enkephalin), biogenic amines: (serotonin, histamine, dopamine, norepinephrine, adrenaline), peptides of the renin-angiotensin system, angiotensin, angiotensin, angiotensin, vasopressin).

However, the percentage of detection of patients is not always satisfactory.

The present invention solves the problem of finding new diagnostic markers for determining addiction diseases, allowing to increase the accuracy of this assessment by increasing the percentage of identifying people who have a deviation of this indicator from the norm in one direction or another.

The problem is solved using the kit for enzyme-linked immunosorbent assay of antibodies to endogenous bioregulators in blood serum for the detection of addiction diseases by two-stage enzyme-linked immunosorbent assay in human serum in vitro, which is a kit in one box containing at least one 96-well collapsible stripped a tablet with sequentially all conjugates from the series applied onto its surface: β-endorphin, serotonin, dopamine, histamine, orphanine and angiotensin; as well as containers containing positive control samples of human blood serum containing antibodies to the conjugates listed above, negative control samples of human blood serum containing no antibodies to the above compounds, anti-species antibodies against human immunoglobulins labeled with horseradish root peroxidase, phosphate-buffered saline containing tween-20, a substrate buffer solution, a solution of a substrate of tetramethylbenzidine and a stop reagent.

The detection of addiction diseases is based on the interaction of antibodies to endogenous bioregulators (β-endorphin, serotonin, dopamine, histamine, orphanin, angiotensin) contained in the analyzed sample with the antigen of the indicated endogenous bioregulator immobilized on the inner surface of the tablet. The resulting antigen-antibody complex is detected using a conjugate of antibodies against human immunoglobulins labeled with horseradish peroxidase. The results are taken into account by changing the color of the substrate-chromogenic mixture.

Our studies have found that with the development of the disease, addiction, including: toxico-chemical dependence: alcoholism, drug addiction, substance abuse; food addiction, gambling (gambling), the most severe changes occur in the metabolism of key compounds such as β-endorphin, serotonin, dopamine, histamine, orphanin and angiotensin, which are accompanied by a violation of the production of specific natural antibodies. If changes in the content of antibodies to them have occurred, anyway, when: in childhood, at birth, as a result of illness, trauma, stress, with the participation of a mental or toxic, genetic factor, we can identify these changes and talk about the biological factor for the development of addiction disease . The correlation between a change in this level and a clinically confirmed diagnosis allows the use of the claimed kit to identify a number of addiction diseases, including: alcoholism, drug addiction, substance abuse; food addiction, gambling (gambling), as a result of a single blood sampling with increased indicators of sensitivity and specificity of the method and, thus, tells the whole invention the criterion of "industrial applicability".

Thus, the technical result of the invention is to increase the accuracy of determining the disease of dependence by increasing the sensitivity and specificity of the method by using a set of 6 markers.

From the prior art, we do not know information regarding the simultaneous detection of the development of a number of addiction diseases in the analysis of antibodies to endogenous bioregulators, as stated in the SET, which are determined in a patient in one blood sample.

Thus, the established by us regularity informs the entire invention both according to the criterion of "novelty" and the criterion of "inventive step".

Table 1 shows the minimum content of antibodies to β-endorphin, serotonin, dopamine, histamine, orphanin, angiotensin, determined using the kit.

Table 2 shows the OD450 values in ELISA on average for groups of 1000 people with alcoholism, opiate addiction and donors when using Orfanin as a hapten, where * p <0.01 (in comparison with donors).

Table 3 shows the values for calculating the sensitivity and specificity of the method when using the inventive kit and one of its components (orphanin)

Equipment and reagents necessary when working with the kit:

- Spectrophotometer vertical scan, which allows to measure the optical density of the solution in the wells of the tablet at a wavelength of 450 nm;

- dry-air thermostat, allowing to maintain a temperature of + 37 ± 0.1 ° C;

- pipettes semi-automatic single-channel with a variable volume with interchangeable tips, allowing you to select volumes of liquid 2-20 μl, 20-200 μl, 200-1000 μl;

- a semi-automatic eight-channel pipette with a variable volume with interchangeable tips, which allows you to select a liquid volume of 50-200 μl;

- graduated cylinders with a capacity of 50 and 100 ml;

- volumetric flask with a capacity of 200 ml;

- glass bottles with a capacity of 5.0 ml;

- baths for reagents or glass Petri dishes;

- filter paper;

- distilled water;

- rubber or plastic gloves

The potential risk of using the kit is class 2a.

All components of the kit, with the exception of the stop reagent (sulfuric acid solution), are not toxic in the concentrations used.

The sulfuric acid solution has an irritating effect. If it comes into contact with the skin and mucous membranes, wash it off with plenty of running water.

Example 1. A kit containing a tablet, individual strips of which are sensitized by six different antigens (β-endorphin, serotonin, dopamine, histamine, orphanine, angiotensin).

The kit includes:

- a 96-well collapsible strip plate (consisting of 12 strips, each of which has 8-holes with designations A through H). with immobilized conjugated β-endorphin antigens (strips No. 1-2), serotonin (strips No. 3-4), dopamine (strip No. 5-6), histamine (strip No. 7-8), orphanine (strip No. 9-10) and angiotensin (strips No. 11-12) - 1 pc.;

- positive control sample No. 1 (human serum containing antibodies to β-endorphin), concentrated - 1 vial. (0.05 ml);

- positive control sample No. 2 (human blood serum - containing antibodies to serotonin), concentrated - 1 vial. (0.05 ml);

- positive control sample No. 3 (human serum containing antibodies to dopamine), concentrated - 1 vial. (0.05 ml);

- positive control sample No. 4 (human serum containing antibodies to histamine), concentrated - 1 vial. (0.05 ml);

- positive control sample No. 5 (human serum containing antibodies to orphanine), concentrated - 1 vial. (0.05 ml);

- positive control sample No. 6 (human serum containing antibodies to angiotensin), concentrated - 1 vial. (0.05 ml);

- negative control sample No. 1 (human serum that does not contain antibodies to β-endorphin), concentrated - 1 vial. (0.05 ml);

- negative control sample No. 2 (human serum that does not contain antibodies to serotonin), concentrated - 1 fl. (0.05 ml);

- negative control sample No. 3 (human serum that does not contain antibodies to dopamine), concentrated - 1 vial. (0.05 ml);

- negative control sample No. 4 (human serum that does not contain antibodies to histamine), concentrated - 1 fl. (0.05 ml);

- negative control sample No. 5 (human serum that does not contain antibodies to orphanine), concentrated - 1 vial. (0.05 ml);

- negative control sample No. 6 (human serum that does not contain antibodies to angiotensin), concentrated - 1 vial. (0.05 ml);

- conjugate of antibodies against human immunoglobulins conjugated with horseradish root peroxidase, concentrated liquid - 1 vial. (0.5 ml);

- phosphate-saline buffer solution, concentrated, containing tween-20 (FSB-T) - 2 vials. (10 ml each);

- substrate buffer solution, concentrated - 1 vial. (1.5 ml);

- a solution of the substrate tetramethylbenzidine (TMB) - 1 fl. (3.5 ml);

- sulfuric acid (stop reagent), 10% - 1 vial. (10 ml).

Preparation of reagents for analysis

Dissolution of the components should be carried out at room temperature (+ 18-25 ° C) and periodic stirring.

To prepare a buffer solution, the contents of one bottle with a phosphate-salt buffer concentrate (FSB-T) are transferred to a 200 ml volumetric flask, rinsed twice with distilled water, all washings are combined with diluted FSB-T, and the total volume is adjusted to the mark with distilled water.

Preparation of positive control solutions produce by adding 0.025 ml of the corresponding concentrated positive control sample of antibodies of the corresponding endogenous bioregulator to 0.225 ml of diluted FSB-T.

Preparation of Negative Control Samples produced by adding 0.025 ml of the corresponding concentrated negative control sample that does not contain antibodies of the corresponding endogenous bioregulator to 0.225 ml of diluted FSB-T.

Preparation of Anti-Antibody Conjugate Solution human immunoglobulins are carried out by adding 2.0 ml of diluted FSB-T to the vial and adding 0.02 ml of a concentrated conjugate of antibodies to human immunoglobulins to it.

A substrate-chromogenic mixture is prepared immediately before use by adding 0.2 ml of a concentrated substrate buffer solution diluted with 1.8 ml of distilled water to 0.5 ml of a TMB substrate solution.

Stop reagent is ready to use.

Tablet preparation

0.2 ml of diluted FSB-T is added to all wells of the plate, stirred by shaking for 5 seconds and the solution is removed by decantation. Washing is repeated two more times. After the last wash, the remaining liquid is removed from the wells by tapping the plate in an inverted position on filter paper.

All blood serum samples are diluted 100 times with diluted FSB-T:

Example 2. The analysis:

The introduction of a negative control sample.

0.1 ml of negative control sample No. 1 was added to the wells of plate A1, A2.

0.1 ml of negative control sample No. 2 was added to the wells of plate A3, A4.

In the wells of tablet A5, A6 contribute 0.1 ml of a negative control sample No. 3

In the wells of the tablet A7, A8 contribute 0.1 ml of a negative control sample No. 4.

In the wells of tablet A9, A10 contribute 0.1 ml of a negative control sample No. 5.

In the wells of tablet A11, A12 contribute 0.1 ml of a negative control sample No. 6

The introduction of a positive control sample.

In the wells of tablet B1 and B2 contribute 0.1 ml of a positive control sample No. 1

0.1 ml of positive control sample No. 2 was added to the wells of plate B3 and B4.

0.1 ml of positive control sample No. 3 were added to the wells of plate B5 and B6.

0.1 ml of positive control sample No. 4 was added to the wells of plate B7 and B8.

0.1 ml of a positive control sample No. 5 were added to the wells of plate B9 and B10.

0.1 ml of positive control sample No. 6 was added to the wells of plate B11 and B12.

For the remaining wells of a collapsible tablet consisting of strips sensitized with various panel antigens mentioned above, add β-endorphin, serotonin, dopamine, histamine, orphanin and angiotensin for each of the antigens in duplicates of 0.1 ml of the studied blood serum samples. The test samples of blood serum and incubated for 1 h at a temperature of + 37 ° C. Then the contents of all wells of the plates are removed by decantation, the wells are washed, and 0.1 ml of a solution of the conjugate of antibodies against human immunoglobulins is added to them. After 1 h, the contents of the wells of the tablets are removed and washed as described above. Then 0.1 ml of substrate-chromogenic mixture prepared 15 minutes before the end of the second incubation is added to all wells of the plates. The plates are kept at room temperature (+ 18-25 ° C) in a dark place for 5-10 minutes, depending on the degree of color development, and 0.05 ml of a stop reagent solution are added to them.

The measurement of optical density (OD) in the wells of the tablets is carried out at a wavelength of 450 nm. Staining the contents of the tablet is stable for no more than 1 hour at room temperature in a dark place.

Accounting for results.

Based on the results of ELISA, the content of class M immunoglobulins (IgM) was analyzed in individual sera of patients and healthy donors, determining a significant change in the level of antibodies.

The test sample is regarded as serum with a normal content of antibodies to endogenous bioregulators if the average optical density in the wells with the analyzed sample corresponds to the value calculated by the formula:

OPnorm = OPotr ± 0.20,

where OPnorm is the average optical density in the well with the analyzed sample,

OPotr is the average optical density in a well with a negative control sample (based on a survey of a group of donors, the average optical density varies from 0.55 to 0.65).

The test sample is regarded as serum that differs from the norm in the content of antibodies to endogenous bioregulators, if the optical density in the wells with the analyzed sample is lower than 0.35 or higher than 0.85.

Example 3. ELISA determination of antibodies to β-endorphin, serotonin, dopamine, histamine, orphanin and angiotensin, where * p <0.01 when examining healthy individuals.

When examining healthy individuals (a group of 1000 people) without a diagnosis of addiction, the following percentage distribution of the presence of antibodies in the blood serum to the studied antigen panel was established.

For dopamine, in 7% of patients, an excess of 0.85 of the optical density value is exceeded, and in 12% of patients this parameter is below 0.35 of the optical density value. For other antigens, the following pattern was revealed. For serotonia, 15% of the examined had an excess, and for 6% a decrease in these indicators. The following distribution was observed for β-endorphin and orphanin: 16% and 15% of people had antibody levels above 0.85, and in 14% and 11% of the examined, the levels of antibodies were below 0.35. For histamine and angiotensin, these values were 11% and 8% increase and 14% and 9% decrease, respectively.

Thus, in the group of examined healthy people there is a population with an increased and decreased level of antibodies to the studied antigens. It is these people who represent the risk group prone to disease addiction, because in most patients, during their examination, we found exactly the same indicators.

The minimum content of antibodies to β-endorphin, serotonin, dopamine, histamine, orphanin, angiotensin, determined using the kit, is shown in table 1 and is not more than 3-6 μg / ml.

For a practical assessment of the results obtained for the clinic and the possibility of their use in the future as an additional diagnostic criterion, a comparison of the results of ELISA of the determination of natural antibodies with URP - the level of rehabilitation potential was carried out. The results indicate a correlation between the various forms of addiction diseases, determined using the proposed set, and determined using URP.

Example 4. An enzyme immunoassay (ELISA) for the determination of antibodies to orphanin in human serum.

Based on the results of ELISA, a comparative analysis of the content of class M immunoglobulins (IgM) in individual sera of patients and donors was carried out, determining a significant change in the level of antibodies (see table 2).

As can be seen from the table, for patients with opiate addiction, the difference in the optical density of the analyzed sample and the reference value of the optical density of the blood samples of healthy donors is positive. These data are confirmed by the clinical diagnosis in 95% of the examined.

For patients with alcoholism, this difference has negative values. These data are confirmed by a clinical diagnosis in 94% of the examined.

For patients with gambling addiction, the group average optical density value in the analyzed samples was significantly reduced compared to the optical density value of the reference blood serum samples of healthy donors. These data are confirmed by the clinical diagnosis in 97% of the examined.

For patients with substance abuse, this difference is positive and is confirmed by the clinical diagnosis in 92% of the examined.

- Example 5. ELISA determination of antibodies to β-endorphin, serotonin, dopamine, histamine, orphanin and angiotensin, where * p <0.01 when examining patients with opium addiction.

- When examining the level of antibodies in a group of 1000 patients with opium addiction, the group average exceeded 0.85 of the optical density for dopamine at 92%, for orphanine at 95%, for β-endorphin at 91%, for serotonin at 87% patients. The content of antibodies to histamine and angiotensin was in the range from 0.35 to 0.85 optical units.

Thus, the initial diagnosis of opium addiction - was confirmed using the inventive set to change the level of antibodies using 4 markers

Example 6. ELISA determination of antibodies to β-endorphin, serotonin, dopamine, histamine, orphanin and angiotensin, where * p <0.01 when examining patients with alcohol dependence.

- In the study of the level of antibodies in a group of 1000 patients with alcohol dependence, a decrease in the index of 0.35 of the optical density value for orphanine in 94%, for β-endorphin in 96%, for angiotensin in 81%, for dopamine in 80%, was established in the group average , patients. The content of antibodies to serotonin, histamine was in the range from 0.35 to 0.85 optical units.

Thus, the initial diagnosis - alcohol dependence - was confirmed using the inventive kit to change the level of antibodies using 4 markers

Example 7. ELISA determination of antibodies to β-endorphin, serotonin, dopamine, histamine, orphanin and angiotensin, where * p <0.01 when examining patients with substance abuse.

- When examining the level of antibodies in a group of 1000 patients with substance abuse, it was found that the group average exceeds 0.85 of the optical density for dopamine in 95%, for serotonin in 81%, for histamine in 94% of patients, a decrease in the 0.35 optical value densities for orphanin at 90%, for β-endorphin at 91%. The content of antibodies to angiotensin was in the range from 0.35 to 0.85 optical units.

Thus, the initial diagnosis of substance abuse was confirmed using the inventive kit to change the level of antibodies using 5 markers.

Example 8. ELISA determination of antibodies to β-endorphin, serotonin, dopamine, histamine, orphanin and angiotensin, where * p <0.01 when examining patients with gambling (gambling).

- When studying the level of antibodies in a group of 1000 patients with gambling addiction, a decrease in the index of 0.35 of the absorbance value for β-endorphin in 97% for orphanine in 91% was found in the group average, an increase in the 0.85 absorbance value for serotonin in 81 %, for dopamine in 80% of patients. The content of antibodies to angiotensin, histamine was in the range from 0.35 to 0.85 optical units.

Thus, the initial diagnosis of gambling - was confirmed using the inventive kit to change the level of antibodies using 4 markers.

Example 9. ELISA determination of antibodies to B-endorphin, serotonin, dopamine, histamine, orphanin and angiotensin, where * p <0.01 when examining patients with food dependence.

- When examining the level of antibodies in a group of 1000 patients with food dependence, it was found that the group average exceeds 0.85 optical density for dopamine in 85%, for serotonin in 80%, for histamine in 89% of patients, a decrease in 0.35 optical density for β-endorphin in 79%. The content of antibodies to orphanin, angiotensin was in the range from 0.35 to 0.85 optical units.

Thus, the initial diagnosis - food dependence - was confirmed using the inventive kit to change the level of antibodies using 4 markers.

Calculation of the sensitivity and specificity of the method

The results of the study of blood serum samples of patients with various addiction diseases, obtained using reagents, were compared with the results of the diagnostic report of a specialist doctor, recorded in the medical history of each tested patient. Based on the data obtained, the sensitivity and specificity of the method were calculated. Moreover, in each group the number of patients with false positive, false negative and truly positive and negative results was determined. The calculation was carried out according to the formulas.

1 Number of true positive test results.

3 Number of false positive test results.

4 Number of false negative test results.

.5 Sensitivity Method: $$\text{H}=\frac{\text{IP}}{\text{IP}+\text{LO}}\cdot \text{one hundred\%}$$

.

.6 Specificity of the method: $$\text{FROM}=\frac{\text{AND ABOUT}}{\text{AND ABOUT}+\text{LP}}\cdot \text{one hundred\%}$$

.

When using the panel of antigens that make up the Kit, the following results were obtained (see table 3). For comparison, the table shows the results of the calculation of similar indicators when using only one marker (orphanin).

As can be seen from the above data, when examining patients with various addiction diseases, we obtain the sensitivity and specificity of the enzyme-linked immunosorbent assay for determining antibodies to endogenous bioregulators in the blood serum using the claimed kit exceeding those obtained using separate endogenous bioregulators.

Thus, the above examples confirm that the proposed spectrum of reagents for enzyme-linked immunosorbent assay of antibodies to endogenous bioregulators in blood serum allows to identify the changes in the level of antibodies in various forms of painful dependencies with high accuracy (reliability), while the use of only one or several markers from the listed set may lead to a distortion of the diagnosis.

Table 1 Sensitivity of determination of antibodies to β-endorphin, μg / ml, no more 3.0 Sensitivity of determination of antibodies to serotonin, mcg / ml, not more than 4.0 Sensitivity of determination of antibodies to dopamine, mcg / ml, not more than 3,5 Sensitivity of determination of antibodies to histamine, mcg / ml, not more than 5,0 Sensitivity of determination of antibodies to orphanin, mkg / ml, no more 4.0 Sensitivity of determination of antibodies to angiotensin, mcg / ml, not more than 6.0

table 2 Group of patients and donors The value of OD ₄₅₀ Orfanin in ELISA on average for the group Number of people with a confirmed diagnosis% Opiate addicts 1.34 ± 0.056 95 Alcoholics 0.43 ± 0.065 94 Game addicts 0.31 ± 0.050 97 Food Addiction Patients 0.61 ± 0.071 Not determined Substance Abuse Patients 0.39 ± 0.070 92 Donors (reference) 0.59 ± 0.021 σ = 0,029 - p <0.01 (in comparison with donors).

Table 3 Addiction diseases Opium addiction Alcohol addiction Game addiction Toxic
niya Food addiction The sensitivity of the method when using the inventive kit,% 98 95 95 96 95 The specificity of the method when using the inventive kit,% one hundred one hundred one hundred one hundred one hundred The sensitivity of the method when using orphanin,% 95.1 93.8 90.2 94.2 Not determined The specificity of the method when using orphanin,% 93.2 92.5 93.7 95.1 Not determined

Claims (1)

A set of reagents for enzyme-linked immunosorbent assay of antibodies to endogenous bioregulators in blood serum for the detection of addiction diseases by the method of two-stage enzyme-linked immunosorbent assay in human serum in vitro, which is a kit in one box containing at least one 96-well collapsible stripped plate coated with its surface is consistently with all conjugates from the series: β-endorphin, serotonin, dopamine, histamine, orphanine and angiotensin; as well as containers containing positive control samples of human blood serum containing antibodies to the conjugates listed above, negative control samples of human blood serum containing no antibodies to the above compounds, anti-species antibodies against human immunoglobulins labeled with horseradish root peroxidase, phosphate-buffered saline a solution containing tween-20, a substrate buffer solution, a solution of a substrate of tetramethylbenzidine and a stop reagent.