RU2557936C2 - Method for increasing diagnostic effectiveness of immunochromatographic systems of pathogen detection - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method is based on contacting a membrane test strip with an analysed fluid sample and initiating thereby a motion along the test strip membranes of reagents being parts of the sample or coating the membrane, and forming the immune complexes to be detected in the course of reactions in the membrane pores or on the surface thereof. A distinguishing feature of the presented method for antigen detection is that the test strip is coated additionally within the test sample contact area with a certain amount of specific antibodies, which react to the detected antigen expected to be found in the sample, when a fluid front moves and block a certain number of binding sites. The number of the coating free antibodies is specified so that the low content thereof in the analysed sample being of no diagnostic importance ensures blocking the binding sites completely that prevents the antigen from binding in the analysed area of the test strip and from developing a destructive staining in the analysed area.

EFFECT: presented approach enables reliable diagnosing based on the detection results of the antigens of gastrointestinal disorders, avoiding the achievement of positive test results for the low-antigen samples, which testifies to no development of the disease in an individual.

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Description

The invention relates to immunology and medical diagnostics and is a method of immunochromatographic analysis. The presence of antigens in the stool samples (pathogenic microorganisms, viruses and markers) is an effective criterion that makes it possible to diagnose the corresponding infectious disease with high reliability. The advantages of this approach, especially for the diagnosis of diseases of the gastrointestinal tract, are non-invasiveness of the sampling, low cost and high reliability of the results. Although both microbiological and immunological methods for the diagnosis of infectious diseases are actively used at present, for conducting mass primary screening, it is optimal to determine the presence of detectable diagnostically significant antigens in stool samples, which can be realized with high expressivity and productivity. Of particular interest are immunochromatographic approaches in those cases where, due to the characteristics of the growth of microorganisms, obtaining the results of microbiological testing may require considerable time.

Worldwide, gastrointestinal disturbances, through infection or toxic poisoning, are one of the leading causes of morbidity and mortality. The World Health Organization estimates that disruption of the gastrointestinal tract causes about 25% of all post-neonatal deaths (Bryce J et al., 2005). Problems associated with impaired bowel function are currently observed in 20% of the population in developed countries. In third world countries, they are responsible for many deaths, and in developed countries they are the main cause of economic losses. In most acute and chronic cases of disorders of the gastrointestinal tract, the pathogen remains unknown, and the only treatment option is maintenance therapy, symptomatic treatment, and diligent attention to nutrition.

Accurate and timely diagnosis is the basis of all drug therapy, but at the same time, modern opportunities for the diagnosis of intestinal infections remain limited, outdated and expensive.

Due to the rapidity, sufficiently high sensitivity and specificity, immunochromatographic tests are indispensable for mass examinations. Immunochemical analysis can be implemented in various formats. However, since the speed and productivity of testing are of primary importance for mass examinations, in this situation immunochromatographic analysis has obvious advantages.

A feature of immunochromatographic test systems is that all reagents necessary for analysis are preliminarily applied to the membrane components of the test strip and its contact with the test sample directly initiates the movement of the liquid front across the membranes, the occurrence of specific reactions and the formation of immune complexes. These complexes, due to the inclusion of a colored marker in their composition, can be detected visually or using an optical detector.

The general scheme of immunochromatography is as follows.

A sample that potentially contains the analyzed antigen, moves under the action of capillary forces along the test strip. Moreover, it first interacts with colored particles (colloidal gold or another marker), on the surface of which specific antibodies are adsorbed. Then, the front of the liquid overcomes the analytical (test) zone, which is a portion of the membrane immobilized with specific antibodies to another epitope of the defined compound. The degree of marker binding in the analytical zone, respectively, the intensity of membrane staining is determined by the concentration of antigen in the sample. To check the quality of the reagents and preserve the functionality of the test system, the control zone located below is used, in which the component adsorbed on the colored particle binds to the corresponding reagent immobilized on the membrane (for example, anti-species antibodies).

The effectiveness of the immunochromatographic test system as a diagnostic tool largely depends on which immunoreagents are used in it and what complexes they form. This applies to the selection of both antibodies and the format of the analysis.

Below is a description of the method for immunochromatographic determination of specific antibodies to Helicobacter pylori, implemented in the test system "CerTest N. pylori test" company "CerTest" (Spain). This technique is considered in this application as a prototype.

CerTest H. pylori is an immunochromatographic test for the qualitative detection of Helicobacter pylori in feces. In the process of analysis, bacterial particulate antigens in a dissolved stool sample react with a stained conjugate (monoclonal antibody-stained latex particles) pre-dried on the membrane of the test strip. Under the action of capillary forces, the mixture moves along the membrane. In the case of a positive result, the conjugate of the colloidal marker conjugates with antibodies, antigen and specific antibodies immobilized on the membrane, in the analytical zone, as a result of which a red line appears. The absence of this line indicates a negative result. At low antigen concentrations, a colored band does not form in the test area. The unbound conjugate interacts with the reagent in the control zone of the test device, forming a colored strip, which indicates the correct conduct of the test.

A significant factor preventing reliable diagnosis of diseases in all cases is the presence of a certain amount of antigen in stool samples in the absence of the disease. The reasons for this may be bacterial carriage and contact with non-pathogenic mycobacteria. In this regard, doctors introduce a threshold level of detectable antigen, the excess of which is the basis for the conclusion about the presence of an infectious process, and failure to achieve a high degree of certainty makes a conclusion about the absence of the disease.

However, control of exceeding the threshold level is easily implemented only for systems of quantitative analysis, such as microplate enzyme-linked immunosorbent assay with photometric recording of results. Such diagnostics require the use of special equipment for washing tablets and final photometry, is coupled with the use of a number of reagents, and therefore significantly less effective as a means of high-throughput mass screening compared with immunochromatography. In the case of immunochromatography, the distinction between samples with antigen concentrations above and below the established threshold level for staining brightness in the analytical zone becomes an exclusively subjective decision that reduces the reliability of the results.

To overcome this limitation, an immunochromatographic assay of Helicobacter pylori is proposed in which the binding of a sample antigen in the assay zone is prevented by the addition of a standard amount of specific antibodies that interact with the assay until it reaches the assay zone. Thus, the diagnostic conclusion becomes based on the difference not between a less and more vivid staining, but between its absence (all specific antigens are blocked by antibodies) and presence (there is a lot of antigen, and there are not enough antibodies in the drug used to block it completely).

The effectiveness of the approach proposed in the patent is confirmed by the example below.

Example 1. Determination of Helicobacter pylori by immunochromatographic method

For the manufacture of the test system, a set of mdi Easypack membranes from Advanced Microdevices (India) was used, including a CNPC-SN12 L2-P25 working membrane (pore size 15 μm), a PT-R5 conjugate substrate, and a GFB-applied sample membrane. R4, AP 045 adsorbent membrane and MT-1 laminating protective film.

The following reagents were applied to the membranes:

1. The conjugate of colloidal gold with an average particle diameter of 30 nm and specific antibodies 2 in a mixture with free specific antibodies 1.

2. Specific antibodies to an excellent epitope of a detectable antigen - the analytical zone of the test strip (antibody 1).

3. Anti-mouse antibodies against mouse immunoglobulins - control zone of the test strip.

On 1 cm of the strip, 2 μl of a solution of specific antibodies (1.0 mg / ml) and 2 μl of a solution of antispecies antibodies (0.5 mg / ml) in 50 mM phosphate buffer, pH 7.4 were applied. A colloidal gold conjugate with specific antibodies was applied at a dilution corresponding to D ₅₂₀ = 2.0, in a volume of 8 μl per 1 cm of band, and an antibody solution of 0.02 μg per 1 cm of band was added. For the application of reagents, an IsoFlow dispenser from Imagene Technology (USA) was used. Membrane sheets coated with immunoreagents were cut into individual test strips 4 mm wide.

Immunochromatographic analysis was performed at room temperature. The test strip was immersed in the sample for 1 min in a vertical position, and then removed and placed on a horizontal surface. Colloidal gold binding was detected after 10 min visually or by receiving a digital image of the test strip using a scanner.

Example 2. Determination of Helicobacter pylori by immunochromatographic method

For the manufacture of the test system, a set of mdi Easypack membranes from Advanced Microdevices (India) was used, including a CNPC-SN12 L2-P25 working membrane (pore size 15 μm), a PT-R5 conjugate substrate, and a GFB-applied sample membrane. R4, AP 045 adsorbent membrane and MT-1 laminating protective film. The following reagents were applied to the membranes:

4. The conjugate of colloidal gold with an average particle diameter of 30 nm and specific antibodies 2 in a mixture with free specific antibodies 1.

5. Specific antibodies for an excellent epitope of a detectable antigen - the analytical zone of the test strip (antibodies 2).

6. Antiviral antibody preparation against mouse immunoglobulins - control zone of the test strip.

On 1 cm of the strip, 2 μl of a solution of specific antibodies (1.0 mg / ml) and 2 μl of a solution of antispecies antibodies (0.5 mg / ml) in 50 mM phosphate buffer, pH 7.4 were applied. A colloidal gold conjugate with specific antibodies was applied at a dilution corresponding to D ₅₂₀ = 2.0, in a volume of 8 μl per 1 cm of band, and an antibody solution of 0.02 μg per 1 cm of band was added. For the application of reagents, an IsoFlow dispenser from Imagene Technology (USA) was used. Membrane sheets coated with immunoreagents were cut into individual test strips 4 mm wide.

Immunochromatographic analysis was performed at room temperature. The test strip was immersed in the sample for 1 min in a vertical position, and then removed and placed on a horizontal surface. Colloidal gold binding was detected after 10 min visually or by receiving a digital image of the test strip using a scanner.

A test was conducted of a sample of 20 samples of feces of patients and 20 individuals without symptoms of the disease (see Table 1). Staining in the analytical zone was revealed for 18 patients and 1 healthy subject, i.e. diagnostic efficiency of the test system exceeds 90% (for traditional analytical systems, this value varies from 50 to 90%), these results were achieved both when implementing the analysis scheme in Example 1, and in the analysis scheme based on Example 2.

Table 1 The results of a comparative test of the proposed immunochromatographic system for the qualitative determination of specific antibodies and enzyme-linked immunosorbent assay Surveyed group Number of persons A positive result according to immunochromatography A negative result according to immunochromatography The presence of antibodies in concentrations above 0.1 mg / ml according to ELISA The presence of antibodies in a concentration not exceeding 0.1 mg / ml, according to ELISA The absence of antibodies according to ELISA Tuberculosis patients twenty eighteen 2 twenty 0 0 Persons without symptoms twenty one 19 one four fifteen

Claims (1)

A method for determining pathogenic microorganisms, including immunochromatographic analysis, in which complexes are formed on the membrane test strip, which include antibody molecules 1 and 2, specific for different epitopes of the microorganism antigen, in which specific antibodies 1 are immobilized in the analytical zone of the test strip, the antigen is contained in the test liquid sample, and specific antibodies 2 are immobilized on colloidal gold particles; also, a fixed amount of free specific antibodies 1 or 2 are applied to the test strip, which interact with the detected antigen when moving along the membrane of the test liquid sample.