RU2846805C1 - Immunochromatographic test system for noninvasive determination of the level of antiovarial antibodies in biological fluid and a method for determining antiovarial antibodies using a test strip included in the test system - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: an immunochromatographic test system is proposed, which is a non-invasive tool for the diagnosis of autoimmune oophoritis, including a test strip for immunochromatographic analysis of biological fluids, excluding blood. A method for determining the presence of antiovarial antibodies in a test sample of biological fluid using the specified immunochromatographic test system is also proposed.

EFFECT: invention makes it possible to quickly and reliably diagnose autoimmune oophoritis by determining the level of antiovarial antibodies in a biological fluid.

10 cl, 1 dwg, 1 tbl

Description

The first invention relates to means for diagnosing the level of antiovarian antibodies in a biological fluid.

Autoimmune oophoritis (hereinafter referred to as AO) is an organ-specific autoimmune disease, the pathogenesis of which is driven by antiovarian antibodies to steroid-producing ovarian cells. The resulting AG-AT immune complexes lead to hyper- or normogonadotropic insufficiency and suppression of folliculogenesis.

The challenge facing the inventors is to create a convenient, non-invasive, and accessible tool that allows for the rapid and highly accurate determination of the presence of antiovarian antibodies in human biological fluids.

Saliva is the predominant biological fluid chosen for testing.

Saliva has great potential as a diagnostic fluid, as it can be used as a biological material for monitoring the general condition of the body, early diagnosis of diseases, and monitoring the effectiveness of therapy.

Saliva is a mixture of secretions from the major and minor salivary glands, mucosal secretions, gingival fissure fluid, serum and blood derivatives from oral wounds, desquamated epithelial cells, bronchial and nasal secretions, bacteria and their metabolic products, viruses and fungi, other cellular components, and food debris. It is a multicomponent system containing a full range of hormones, proteins, enzymes, antibodies, antimicrobial components, and cytokines, which are absorbed into saliva from the blood. Therefore, in many cases, it is possible to use salivary biomarkers instead of blood biomarkers.

The main advantage of using saliva for these purposes is the simplicity and non-invasiveness of collection.

LEVEL OF TECHNOLOGY

A diagnostic tool for AO is known from the prior art, which is an immunochromatographic test system “Fertility Test Kit No. 20152401287” (Beijing Zhongjian Antai Diagnosis Technology Co., Ltd, China)

The disadvantage of this tool is the use of blood serum as a diagnostic material, as well as the possibility of detecting only immunoglobulins (Ig) class G without taking into account IgM.

The prior art includes test strips for determining pregnancy, antibodies to the Covid-19 virus, hepatitis D, HIV, etc.

The closest analogue to the proposed test system is the device described in patent No. RU(11) 2800845(13)C2 “METHOD AND MEANS FOR RAPID DETECTION OF HEPATITIS D VIRUS INFECTIONS”, which is characterized by the following features:

"- an immunochromatographic testing device, such as an immunochromatographic assay (ICA) device;

- the immunochromatographic testing device is a point-of-care device;

- the sample for the study is saliva"

The disadvantage of this immunochromatographic testing device is that it cannot be used to diagnose AO.

The technical problem is the lack of technical means that would allow for the non-invasive determination of the presence of antiovarian antibodies in a biological fluid sample with high accuracy and speed.

The technical result of the first claimed solution is the creation of a non-invasive means for diagnosing AO in the form of a test system, including test strips, test cassettes, a buffer solution in a vial, a device for collecting samples of biological fluid with a set of disposable tubes and containers, allowing for the rapid, highly reliable diagnosis of AO by determining the level of antiovarian antibodies in the biological fluid.

The test strip design, which is part of the test system, is based on the diffusion-osmotic movement of the compound being detected (analyte, eluent) in a liquid flow along a nitrocellulose membrane, which is typically manufactured as a thin strip and carries several analyte capture zones (vertically arranged stripes relative to the membrane) necessary for binding the compound of interest from the moving solution. These zones can be represented by molecules complementary to the compound of interest adsorbed on the membrane surface. Thus, specific immune complexes (ICs) are formed in the capture zone region during a specific reaction between the analyte and a known compound bound to the test strip surface. ICs are colorless conglomerates of two or more molecules, typically an antigen and an antibody (Ag-Ab). Therefore, their visualization requires chemicals capable of forming pigments recognizable by the human eye and/or a detecting analyzer. These chemicals are called tracers, and the most common today are enzyme molecules, latex, and metal ions (colloidal gold nanoparticles). On the test strip, the tracers are attached to a fiberglass backing and are conjugated with antibodies at the site of application of the test sample.

There are four layers attached to the test strip backing.

The layer (2) for introducing the sample is a filter and is necessary to create capillary forces that facilitate the movement of the introduced material (9) towards the layer with the conjugate (3).

The conjugate (3) is a mixture of recombinant antigens conjugated with colloidal gold and biotinylated with antibodies to human IgM.

This layer contains antibodies to ovarian tissue antigens, designed to interact with those present in the biological sample, designed to interact with recombinant antigens conjugated with colloidal gold nanoparticles as an indicator, as a result of which, during the process of such fluid migration, an immune complex is formed.

IgM antibodies interact with recombinant antigens conjugated with colloidal gold and biotinylated antibodies to human IgM.

IgG antibodies also interact with recombinant ovarian tissue antigens conjugated with colloidal gold. The resulting immune complexes migrate through a nitrocellulose membrane (7) and are immobilized upon passing through the appropriate test strip.

The test strip has three test lines (4, 5, 6). To form three clearly demarcated lines (control strip M (4), test strip G (5), and test strip C (6)), immobilized control antibodies, antibodies to human IgG, and streptavidin (test strip for detecting IgM antibodies) are applied to the surface of the nitrocellulose membrane.

The IgM immune complex is immobilized in the streptavidin application area (test strip M) on the membrane, resulting in the development of a purple color, indicating a positive result for the determination of class M antibodies.

The IgG immune complex is immobilized in the area of application of antibodies to human IgG (test strip G) on the membrane, resulting in the development of a purple color (a positive result for the determination of IgG antibodies).

The coloration of the control strip C appears when the chicken yolk immunoglobulin IgY conjugated with colloidal gold nanoparticles binds to the control antibodies applied in the control zone.

Immune complexes not fixed on the test lines move towards the adsorbent layer (8), represented by cotton material and necessary for removing excess biological fluid and conjugates.

An immunochromatographic test system for non-invasive determination of the level of antiovarian antibodies in a biological fluid includes a buffer solution, an immunochromatographic test strip, a device for collecting samples of biological fluid with a set of disposable tubes and containers.

Buffer solution - phosphate buffer, pH = 7.4; 20 mM.

An immunochromatographic test strip for non-invasive determination of the level of antiovarian antibodies in biological fluids, excluding blood, (hereinafter referred to as the test strip) is shown in the figure, where

1 - supporting part;

2 - sample application layer;

3 - layer with conjugate;

4 - test line M;

5 - test line G;

6 - control line C;

7 - nitrocellulose membrane;

8 - adsorbent layer;

9 - biological fluid solution.

The test strip contains a nitrocellulose membrane as a base.

The membrane carries three analyte capture zones, located vertically relative to the membrane, and the zones are represented by molecules complementary to the desired compound, adsorbed on the membrane surface:

- zone C contains antibodies similar in structure to a person’s own antibodies, zone M contains a high-affinity protein sensitive to biotinylated human proteins, zone G contains antibodies to human autoantibodies necessary for binding the desired compound from a moving solution;

- where immune complexes are formed in the area of capture zones - the result of a specific reaction of the analyte with compounds adsorbed on the membrane surface that fix antibodies;

- where the immune complex is a colorless conglomerate of two or more molecules, visualized by labels in the form of colloidal gold nanoparticles, which are fixed in the form of conjugates with antibodies on a glass fiber substrate at the site of application.

The antibodies applied to zone C were anti-chicken IgY antibodies at a concentration of 0.09±0.018 μg.

The antibodies applied to the G zone are represented by antibodies to human IgG at a concentration of 0.30±0.06 μg.

The antibodies applied to the M zone are represented by the protein streptavidin at a concentration of 1.2±0.05 μg.

EXAMPLE OF EXECUTING A TEST SYSTEM

Completeness

The test system is a set of reagents, which contains:

1. Test cassette - 25 pcs.;

2. Bottle with buffer solution - 1 pc.;

3. SaliCaps saliva sampling device with a set of disposable tubes and containers - 25 pcs;

4. Instructions for the medical device - 1 pc.

5. Cardboard box.

Does not contain medicinal substances or products of human origin.

The test cassette is represented by 6 components described in Table 1 (see in the graphic section).

The second claimed solution is “A METHOD FOR DETERMINING ANTI-OVARIAN ANTIBODIES USING THE ABOVE-MENTIONED TEST STRIP INCLUDED IN THIS SYSTEM”

The second claimed invention relates to medicine, namely to methods of dry immunochemistry, qualitative identification of the presence of autoantibodies in a biological fluid using test systems that include immunochromatographic test strips for non-invasive determination of the level of antiovarian antibodies in a biological fluid.

The technical problem is the lack of solutions that allow for the qualitative determination of the presence of antiovarian antibodies in an oral fluid sample using immunochromatographic analysis.

The ICA method is known from the prior art and is widely used in modern medical practice as a highly specific and accurate test for detecting hormones, narcotic and medicinal substances and their metabolic products, etc., in blood serum samples.

Also known is the "METHOD FOR EARLY DIAGNOSIS OF AUTOIMMUNE OOPHORITIS IN ADOLESCENT GIRLS WITH NORMOGONADOTROPIC OVARIAN HYPOFUNCTION" (Andreeva V.O. et al., 2014), for which patent RU (11) 2521331 (13)C1 was received. A disadvantage of this proposed method is the use of ELISA to search for the serum fraction of the neopterin protein, which is not a reliable and specific indicator of autoimmune damage to ovarian cells.

The technical result of the claimed solution is achieved by using a sample collection system for analysis that does not destroy the analyte being tested in saliva or alter its concentration. As mentioned previously, the analytes tested in saliva are small, non-polar molecules that can be adsorbed by plastics with similar hydrophobic properties.

This method will enable rapid initial screening diagnostics of autoimmune oophoritis, for example, in an emergency room setting. In complex clinical cases, rapid differential diagnosis is possible, requiring the exclusion or confirmation of autoimmune oophoritis.

Furthermore, monitoring the levels of pathological autoantigens is particularly important during treatment. Fighting AIDs can take many years, and sometimes a lifetime, for a patient.

Monitoring the therapeutic effect requires numerous tests, including the detection of pathological autoantibodies, which requires venous blood sampling. This procedure significantly reduces the patient's quality of life. Therefore, the proposed non-invasive method can be used to monitor the clinical response to therapy for AIDs.

It's important to note that the use of non-invasive methods is appropriate in pediatric practice. Based on recent statistics, it can be concluded that the incidence of AIDs in childhood is steadily increasing. For children, numerous traumatic interventions, including blood sampling to determine autoantibody levels and confirm or rule out AIDs and/or monitor therapeutic response, are much more painful and uncomfortable.

Therefore, the emergence and implementation of non-invasive methods in therapeutic and pediatric practice is a very appropriate solution.

Results are read and interpreted 10-15 minutes after the start of the test. When testing some biological samples with low concentrations of IgM and/or IgG antibodies, the color of the test strips may be faint; however, any result in which a color development is visually observed, even at low intensity, should be interpreted as positive.

DESCRIPTION OF THE METHOD

A method for determining the presence of antiovarian antibodies in a test sample of biological fluid using a test strip included in the test system described above, which consists of adding a buffer solution to the biological fluid collection system, which additionally consists of tubes for collecting and containers for storing biological material that are inactive to the test fluid.

To implement the method, a sample of biological fluid is collected and diluted in a buffer solution, which is then applied to the surface of the sample application layer and a time is allowed for the complete diffusion of the biological fluid solution to the adsorbent layer on the surface of the test strip, where the IgM immune complex is immobilized in the M zone of the test strip containing streptavidin, the IgG immune complex is immobilized in the G zone, and the non-immobilized residue of antibodies in the biological fluid solution is adsorbed in the C zone of the control strip.

Saliva is preferably used as a biological fluid, but vaginal secretions and breast milk can also be used.

Polypropylene is the preferred material for tubes and containers.

RESEARCH CONDUCTED

The description of the implementation of the developed test system was carried out at the laboratory for targeted regulation of intermicrobial interactions in exo- and endomicroecological systems of the Kirov State Medical University (Federal Service for Supervision in Education and Science, state accreditation certificate No. 3318 from 23.01.20 to 23.01.2026) together with the Medical and Biological Center for Biomodeling (MCB) of the Kirov State Medical University in accordance with GOST 33215-2014 and GOST 33216-2014.

Using the developed test system, a comparative analysis of two methods (ELISA and ICA) was conducted as part of a scientific qualification project to assess the sensitivity and specificity of serodiagnosis of the autoimmune marker AO—antiovarian antibodies. Immunological blood testing was performed using ELISA test reagents (IBL International GMBH, Germany).

Oral fluid analysis was conducted using the test system proposed by IH. Blood and oral fluid samples from 20 outbred laboratory mice were used for the study (Group 1: 10 mice with autoimmune oophoritis; Group 2: 10 mice without autoimmune pathology). Autoimmune pathology in laboratory animals was modeled by intraperitoneal administration of an ovarian suspension from intact mice, prepared according to an algorithm for experimental modeling of autoimmune pathology (Patent No. RU2439712C1 "Method for Modeling Autoimmune Oophoritis," V.S. Loginov, 2012).

Blood and saliva from animals used for the experimental comparison of two AO diagnostic methods (ELISA and ICA) were collected at the end of the experiment in sterile Eppendorf tubes (blood) and test tubes using the SaliCap system (saliva). Blood was tested for AOA using the aforementioned ELISA test kit. Saliva was analyzed using the proposed test system. The results of the studies showed that AOA was detected in the blood of Group 1 animals using the ELISA method at titers exceeding 1:320 in 100% (10 animals) of cases (p = 0.00063). The analysis performed using the IH test strip showed similar results: 90% (9 animals) of the saliva from Group 1 animals showed a positive result (p = 0.000024), specifically the appearance of the color of the M and G test lines. The G line was significantly more frequently visible (in 70% of cases, p = 0.000266). Thus, the initial testing of the test system demonstrated reliable results in the qualitative detection of antiovarian antibodies.

Claims (11)

1. An immunochromatographic test system which is a non-invasive means for diagnosing autoimmune oophoritis, comprising a test strip for the immunochromatographic analysis of biological fluids, excluding blood, containing, as a base, a nitrocellulose membrane and an adsorbent layer, where the membrane bears the following layers: a layer for introducing a sample; a layer with a conjugate containing an ovarian tissue antigen conjugated with colloidal gold, chicken IgY conjugated with colloidal gold, and biotinylated antibodies to human IgM, and is divided into three zones: test zone M containing streptavidin, test zone G containing antibodies to human IgG, control zone C containing anti-chicken IgY antibodies. 2. The test system according to paragraph 1, characterized in that the membrane carries three analyte capture zones located vertically relative to the membrane, wherein the zones are represented by molecules complementary to the desired compound, adsorbed on the surface of the membrane. 3. The test system according to paragraph 1, characterized in that the antibodies applied to zone C are represented by anti-chicken IgY antibodies at a concentration of 0.09±0.018 μg. 4. The test system according to paragraph 1, characterized in that the antibodies applied to the G zone are represented by antibodies to human IgG at a concentration of 0.30±0.06 μg. 5. The test system according to item 1, characterized in that streptavidin at a concentration of 1.2±0.05 μg is applied to zone M. 6. A method for determining the presence of antiovarian antibodies in a test sample of biological fluid using an immunochromatographic method, characterized in that it is performed using a test system according to paragraph 1, a system for collecting biological fluid, consisting of tubes for collecting and containers for storing biological material that are inactive to the test fluid, a buffer solution, characterized in that: A sample of biological fluid is collected and diluted in a buffer solution, which is then applied to the surface of the sample application layer, and the time is waited for the complete diffusion of the biological fluid solution to the adsorbent layer on the surface of the test strip, where the IgM immune complex is immobilized in the M zone of the test strip containing streptavidin, the IgG immune complex is immobilized in the G zone, and the non-immobilized remainder of the antibodies found in the biological fluid solution is adsorbed in the C zone of the control strip. 7. The method according to paragraph 6, characterized in that saliva is used as the biological fluid. 8. The method according to paragraph 6, characterized in that vaginal secretion is used as the biological fluid. 9. The method according to paragraph 6, characterized in that breast milk is used as the biological fluid. 10. The method according to paragraph 6, characterized in that polypropylene is used as the material for the tubes and containers.