RU2532352C2 - Method of carrying out immunochromatographic analysis for serodiagnostics - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to immunology and medical diagnostics and represents method of carrying out immunochromatographic analysis for serodiagnostics. Claimed invention is intended for immunochromatographic determination of antibodies to causative agents of infectious diseases or other antigens, for instance, allergens, in liquid samples. Claimed method of identifying antibodies is characterised by the following: solution for sample dilution contains specific antibodies against test strip of antigen (or antigens) immobilised in analytic zone. Used concentration of antibodies in diluting solution is lower than lower limit of determination of antibodies by said method with application of diluting solution, which does not contain specific antibodies, which results coloration of analytic zone of test strip is not observed if specific antibodies are absent in sample. If sample contains specific antibodies, presence of additional quantity of specific antibodies in diluting solution results in enhancement of intensity of coloration of test strip analytic zone.

EFFECT: application of invention makes it possible to register total concentration of specific antibodies in sample and in diluting solution, which results in reduction of limit of analysis identification due to displacement of working range of concentrations, determined by test, into the area of lower values.

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Description

The invention relates to immunology and medical diagnostics and is a method for conducting immunochromatographic analysis for serodiagnostics. The presence in the blood serum of antibodies specific for the causative agent of a certain disease, or other antigen, for example, such as an allergen, is an effective criterion that makes it possible to diagnose the corresponding infectious disease or allergy with high reliability (Reznikova L.S., Epstein-Litvak R.V. , Levi M.I. / Serological research methods for the diagnosis of infectious diseases - M., Medgiz, 1962; Medical Microbiology, Virology and Immunology: A Textbook for Students of Medical Universities / Edited by A.A. Vorobyov. - M .: OOO “Medical News Agency”, 2006). In the case of the diagnosis of infectious diseases, the advantages of this approach compared to the direct identification and identification of the pathogen are certainty when choosing a test sample (blood serum, while the pathogen can be mainly localized in various organs and tissues at this stage of infection), as well as the possibility of quick detection is sufficient high levels of antibodies induced by contact with the antigen, while quite long tadii of growing until they reach a registered concentration. Although both microbiological and immunological methods for diagnosing infectious diseases are currently actively used, for the primary primary screening, the immunological determination of the presence of specific antibodies in the blood serum (serodiagnosis) is optimal, which can be implemented with high expressivity and productivity. Of particular interest are serodiagnostic approaches in those cases where, due to the characteristics of the growth of microorganisms, obtaining the results of microbiological testing may require considerable time. In the case of allergy diagnosis, antibodies against a particular allergen are the main marker of this functional disorder (IgE antibodies in the case of immediate allergies and IgG antibodies in the case of delayed allergies), therefore, laboratory allergy diagnosis methods often come down to detecting antibodies, or identifying the body's reaction associated with the presence of these antibodies (as in the case of a provocative test) (Patterson R., Grammer L.K., Grinberger P.A. Allergic diseases: diagnosis and treatment / Translation from English, ed. Academician of the RAMS A.G. Chuchalina (Ch. ed.), Corresponding Member of the RAMS I.S. Gushchina (ed. Ed.) - M: GEOTAR-Media, 2000). These reasons determine a significant interest in serodiagnostic methods.

Due to the rapidity, sufficiently high sensitivity and specificity, serological tests are indispensable for mass examinations. In addition, the humoral immune response reflects an active infectious process, and therefore the results of immunochemical testing reliably reflect precisely the cases of the disease, discriminating them against bacterial carriage.

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 undoubted advantages, for which all the necessary reagents 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 course of specific reactions and the formation of immune complexes, which, thanks to the inclusion of a colored mark Parameters can be detected visually or with an optical detector (Fig. 1).

In relation to serodiagnosis (determination of antibodies specific to a particular antigen or group of antigens), the general scheme of immunochromatography is as follows.

A sample potentially containing specific antibodies, when in contact with the test strip under the action of capillary forces, moves along the test strip. Moreover, it first interacts with colored particles, on the surface of which a component intended for binding to antibodies is adsorbed (conjugated). Often, anti-species antibodies (immunoglobulins isolated from the serum of an animal immunized with a human immunoglobulin preparation or another organism for which serodiagnosis is performed) are chosen as such a component, protein A from Staphylococcus aureus, protein G from Streptococcus spp. or other reagents for binding antibodies. Colloidal gold is most often used as a label, and as a conjugation method, it is simple physical adsorption of the protein on the surface, followed by separation of the non-adsorbed molecules by precipitation of colloidal gold particles by centrifugation. Then, the liquid front overcomes the analytical (test) zone, which is a portion of the membrane with an immobilized antigen (native or specially modified for effective sorption), as a result of interaction with which complexes of antigen / antigen molecules, specific antibodies and conjugate with colloidal gold are formed . The degree of marker binding to the immobilized antigen and, accordingly, the intensity of membrane staining are determined by the concentration of specific antibodies in the sample. To check the quality of the reagents and maintain the functionality of the test system, the control zone located below is used, in which the component adsorbed on the colored particle is bound to the corresponding reagent immobilized on the membrane.

Below is information about the methodology implemented in the TB-Check-1 test system of Vedalab (France) - see http: // http: //www.sanitamedikal.com/Assets/MD_220002_m3_TB_l408_c.pdf. This method of determining antibodies to the causative agent of tuberculosis is considered in this application as a prototype.

The method is based on a combination of antibodies to human immunoglobulins conjugated to a chromogen and a highly purified BCG protein. When the test sample passes through the adsorption zone of the test device, the conjugate containing labeled antibodies binds to IgG to form an antigen-antibody complex. This complex interacts with a highly purified BCG protein in the test zone of the device and, if the concentration of specific IgG to the tuberculosis pathogen exceeds 350 U / ml, it forms a colored band. At a low concentration of antibodies, 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. Test procedure: fill in a disposable pipette with serum or plasma and add 1 drop to the window for testing the test device. Add 5-6 full drops of dilution solution (diluent) to the sample window. After 10-15 minutes, record the results.

One of the most important tasks in immunochromatography is to increase the sensitivity of the analysis. With regard to serodiagnostics, this means minimizing false negative test results if antibodies in the sample are contained in diagnostically significant concentrations, but below the detection limit of the method. Applicants have proposed an approach to solving this problem, based on adding an additional amount of specific antibodies to the sample at concentrations lower than the detection limit of the method using a dilution solution that does not contain specific antibodies. In this case, the total concentration of antibodies in the sample and the added antibodies may exceed the detection limit and the sample will be diagnosed as positive. Thus, the sensitivity of the method increases due to a shift in the range of determined concentrations to the region of lower values.

The proposed approach was implemented by applicants for serodiagnosis of tuberculosis using the 38 kDa antigen (Ag78, antigen 5, PhoS, Rv0934) M. tuberculosis. The following is a description of the method of obtaining test strips and immunochromatographic analysis, as well as the results.

Example:

To form the test system, we used a set of mdi Easypack membranes from Advanced Microdevices (India), 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. Recombinant antigen 38 kDa M. tuberculosis (Rv0934), the company "Arista Biologicals Inc." (USA), cat. No. AGMTB-0220.

2. The conjugate of colloidal gold with an average particle diameter of 30 nm and a recombinant antigen of 38 kDa M. tuberculosis.

3. Monoclonal antibodies NTM81 against a 38 kD recombinant antigen M. tuberculosis, Center for Molecular Diagnostics and Therapy, Moscow (Russia).

To form the analytical zone, 38 kDa antigen was used, and the control zone was used against 38 kDa antigen. 2 μl of antigen solution (1.0 mg / ml in 50 mM phosphate buffer, pH 7.4) and 2 μl of antibody solution (0.5 mg / ml in the same buffer) were applied to 1 cm of strip. A conjugate of colloidal gold with a 38 kDa antigen was applied in a dilution corresponding to D ₅₂₀ = 2.0, in a volume of 8 μl per 1 cm of the strip. For the application of reagents, an IsoFlow dispenser (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 obtaining a digital image of the test strip using a scanner and quantitatively assessing the color intensity of the analytical zone using the Nonlinear Dynamics TotalLab TL120 v2009 program.

By testing solutions containing fixed concentrations of specific antibodies (NTM81 monoclonal antibodies), a calibration curve was constructed and the limit of visual detection of the method was determined (Fig. 2). Then, blood serum was tested from a patient infected with tuberculosis and from a healthy donor (Fig. 3, A), in this case, no positive test results were observed. After adding an additional amount of specific antibodies to the samples at a concentration corresponding to the limit of visual detection (5 μg / ml), a clear staining of the analytical zone is observed in the serum of the patient (Fig. 3, B). Thus, the proposed approach avoided a false negative test result.

Fig. 1. The principle of immunochromatographic analysis.

Fig. 2. Calibration curve for the determination of specific antibodies against 38 kDa antigen of M. tuberculosis by immunochromatography.

Fig. 3. Testing the blood serum of a patient with tuberculosis and a healthy donor without adding an additional amount of specific antibodies (A) and after adding specific antibodies at a concentration of 5 μg / ml (B).

Claims (1)

A method for conducting immunochromatographic analysis for serodiagnostics, in which an antigen or antigens and colloidal gold particles conjugated with an antibody binding agent are applied to a membrane test strip, characterized in that the sample dilution solution contains specific antibodies against the antigen or antigens used.

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