Method for increasing diagnostic effectiveness of immunochromatographic systems of pathogen detection

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.

1 tbl, 2 ex

 

The invention relates to immunology and medical diagnostics and is a way immunochromatographic analysis. The presence in stool samples of antigens (pathogens, viruses, and markers) is an effective criterion with high reliability appropriate to diagnose infectious disease. The advantages of this approach, especially for the diagnosis of diseases of the gastrointestinal tract, is a non-invasive probe sampling, low cost and high reliability of the results. Although currently actively used both microbiological and immunological methods of diagnosis of infectious diseases, for mass primary screening optimally detect in stool samples determined diagnostically relevant antigens, which can be implemented with high performance and rapidity. Of particular interest are immunochromatographic approaches in cases when the growth of microorganisms obtaining results of microbiological testing may require considerable time.

Worldwide, gastrointestinal disturbance, in the course of infection or toxic poisoning, are a major cause of morbidity and mortality. The world�th health organization estimated, the violation of the gastrointestinal tract is the cause of death in about 25% of all deaths post-neonatal period (Bryce J et al., 2005). The problems associated with dysfunction of the intestine, are currently present in 20% of the population in developed countries. In third world countries they are responsible for many deaths, and in developed countries they are a major cause of economic losses. In most acute and chronic cases of disorders of the gastrointestinal tract pathogen remains unknown, and the only option of treatment is supportive therapy, symptomatic treatment and diligent attention to nutrition.

Accurate and timely diagnosis is the Foundation of all drug therapy, but the possibilities for the diagnosis of intestinal infections remain limited, outdated and expensive.

Thanks to the quick testing were sufficiently high sensitivity and specificity of immunochromatographic tests are indispensable for mass surveys. Immunochemical analysis can be implemented in a variety of formats. However, as for mass screening of high importance speed and performance test, in this situation, the undoubted benefits of immunochromatographic analysis.

Especially�STU immunochromatographic test systems, what all is needed for analysis reagents pre-deposited on the membrane components of the test strip and its contact with the test breakdown directly initiates the movement of a liquid front in the field of membranes, the occurrence of specific reactions and the formation of immune complexes. These complexes due to the inclusion in the composition is colored marker can be detected visually or by an optical detector.

The General scheme of immunochromatography is as follows.

The sample potentially containing the analyte antigen, by capillary action moves along the test strip. While it initially interacts with colored particles (colloidal gold or other marker) on which surface adsorbed specific antibodies. Then the front of the liquid overcomes analytical (test) area, which represents a portion of a membrane with immobilized specific antibodies to another epitope-defined connections. The degree of binding of the marker in the analytical area, respectively, the intensity of staining of the membrane are determined by the concentration of antigen in the sample. For quality control of reagents and maintaining the functionality of the test system used is located next control area in which the component adsorbed on a colored particle, concerned�tsya with the corresponding immobilized on the membrane reagent (for example, antivirovym antibodies).

The efficacy of immunochromatographic test systems as diagnostic tools largely depends on what immunoreagent it uses and which complexes they form. This applies to the selection and antibodies, and format analysis.

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

CerTest N. pylori is a immunochromatographic test for the qualitative detection of Helicobacter pylori in feces. In the analysis of corpuscular antigens of bacterial origin that are dissolved in the stool sample, react with the coloured conjugate (monoclonal antibody-colored latex particles), pre-dried on the membrane test strip. Under the action of capillary forces the mixture moves along the membrane. In the case of a positive result in the analytical area is the binding of colloidal conjugate of a marker antibody, antigen and immobilized on the membrane with specific antibodies, resulting in a line will appear in red. The absence of this line indicates a negative result. At low concentrations of anti�ena colored band in the test zone is formed. Unbound conjugate interacts with the reagent in the control zone of the test device, forming a colored band that indicates the proper conduct of the test.

Significant obstacle to accurate diagnosis of diseases in all cases, is the presence of some amount of antigen in the stool samples in the absence of the disease. Reasons for this may be bacterial carriage and contact with non-pathogenic mycobacteria. In this regard, physicians establish threshold levels of antigen detected, the excess of which is the basis for a conclusion about the presence of infection, and failure allows a high degree of reliability make the conclusion about the absence of disease.

However, the control of a threshold level is easily implemented only for systems quantitative analysis, such as microplate enzyme-linked immunosorbent assay with photometric registration of results. Such a diagnosis requires the use of special equipment for washing tablets and the final photometry involves using a number of reagents and therefore significantly less effective as a means of high-performance mass screening compared with immunochromatography. In the case of immunochromatography discernment of the samples with the concentration of antigen in�above and below the threshold level according to the brightness of staining in the analytical area becomes extremely subjective decision reducing the reliability of the results.

To overcome this restriction in the present application proposed immunochromatographic analysis of Helicobacter pylori, which is binding in the analytical zone of the antigen of the sample is prevented by adding a standard amount of specific antibodies that interact with the sample before reaching her analytical zone. Thus diagnostic assessment is based on the distinction between less and more vivid coloration, and between its absence (all specific antigens blocked by antibodies) and presence (antigen quite a lot, and antibodies used in the drug is not enough to block it completely).

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

Example 1. Definition of Helicobacter pylori immunochromatographic method

For the manufacture of the test system used a set of membranes "mdi Easypack" firm "Advanced Microdevices" (India), comprising a working membrane CNPC-SN12 L2-P25 (pore size 15 μm), the substrate for the conjugate PT-R5, the membrane for application of the sample GFB-R4, absorbent membrane AR 045 and laminating a protective film MT-1.

The membranes were applied the following reagents:

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

2. Specific antibodies on a different epitope of the designated antigen - analytical area of the test strip (antibody 1).

3. The drug antivitamin antibodies against mouse immunoglobulins - a control zone of the test strip.

1 cm strips were applied 2 μl of a solution of specific antibodies (1.0 mg/ml) and 2 μl of a solution antivitamin antibodies (0.5 mg/ml) in 50 mm phosphate buffer, pH 7.4. Conjugate colloidal gold with the specific antibody was applied in a dilution corresponding to D520=2,0, in a volume of 8 μl per 1 cm strips, and added to the antibody solution from the calculation of 0.02 µg per 1 cm of the strip. For application of reagents used dispenser "IsoFlow" company "Imagene Technology (USA). Sheets of membranes with applied immunoreagents was cut into individual test strips 4 mm in width.

Immunochromatographic assay was performed at room temperature. The test strip was immersed into the sample for 1 min in a vertical position, and then removed and placed on a horizontal surface. Detection of binding of colloidal gold was carried out after 10 min or visually receiving a digital image of the test strip with a scanner.

Example 2. Definition of Helicobacter pylori immunochromatographic method

For the manufacture of the test system used a set of membranes "mdi Easypack" firm "Advanced Microdevices" (India), comprising a working membrane CNPC-SN12 L2-P25 (pore size 15 MK�), the substrate for the conjugate PT-R5, the membrane for application of the sample GFB-R4, absorbent membrane AR 045 and laminating a protective film MT-1. The membranes were applied the following reagents:

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

5. Specific antibodies on a different epitope of the designated antigen - analytical area of the test strip (antibody 2).

6. The drug antivitamin antibodies against mouse immunoglobulins - a control zone of the test strip.

1 cm strips were applied 2 μl of a solution of specific antibodies (1.0 mg/ml) and 2 μl of a solution antivitamin antibodies (0.5 mg/ml) in 50 mm phosphate buffer, pH 7.4. Conjugate colloidal gold with the specific antibody was applied in a dilution corresponding to D520=2,0, in a volume of 8 μl per 1 cm strips, and added to the antibody solution from the calculation of 0.02 µg per 1 cm of the strip. For application of reagents used dispenser "IsoFlow" company "Imagene Technology (USA). Sheets of membranes with applied immunoreagents was cut into individual test strips 4 mm in width.

Immunochromatographic assay was performed at room temperature. The test strip was immersed into the sample for 1 min in a vertical position, and then removed and placed on a horizontal surface. Detection of binding of colloidal gold is p�ulali after 10 minutes or visually receiving a digital image of the test strip with a scanner.

Conducted testing 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 area identified for 18 patients and 1 healthy patients, 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 as in the implementation of the scheme of the analysis of example 1 and scheme-based analysis of example 2.

Table 1
The results of comparative tests of the proposed immunochromatographic qualitative determination of specific antibodies and enzyme immunoassay for quantitative analysis
The surveyed groupThe number of peopleA positive result according to immunochromatographyA negative result according to immunochromatographyThe presence of antibodies in a concentration above 0.1 mg/ml according to ELISAThe presence of antibodies at a concentration not exceeding 0.1 mg/ml according to ELISAThe absence of antibodies according to the ELISA
TB patients201822000
Persons without symptoms of disease201191415

Method of determination of pathogens, including immunochromatographic analysis, in which the membrane test strip form a complex, composed of molecules of antibodies 1 and 2, specific towards different epitopes of the antigen of the microorganism, in which 1 specific antibodies immobilized in the analytical zone of the test strip, the antigen contained in a test liquid sample, and 2 specific antibodies immobilized on colloidal gold particles; also on the test strip is applied a fixed number of free specific antibodies either 1 or 2 that interact with a defined antigen when driving on the membrane of the test liquid sample.



 

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FIELD: medicine.

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19 cl, 8 dwg, 3 ex

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1 tbl

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7 cl, 1 ex, 2 dwg

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1 tbl, 2 ex

FIELD: medicine.

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2 ex

FIELD: medicine.

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EFFECT: group of inventions provide high sensitivity and specificity of detection methods.

13 cl, 12 ex, 14 dwg, 12 tbl

FIELD: medicine.

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2 ex

FIELD: medicine, ophthalmology.

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EFFECT: higher accuracy of prediction.

2 ex

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