RU2545909C2 - Method of immunochromatographic determination of specific antibodies - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to immunology and medical diagnostics and represents a method of the immunochromatographic determination of specific antibodies. The method is based on the contact of a membrane test-strip with an analysed liquid sample and initiated by the said contact movement of reagents, contained in the sample or applied on the membrane and forming immune complexes in the course of interactions in the membrane pores or on its surface, on the test-strip membranes. The intensity of colouration of a mark, which bound in the analytical zone, is detected visually. Characteristic peculiarity of the claimed method of the antibody determination consists in the fact that a conjugate of colloid gold with an antigen is used in the test, and the reagent for binding common antibodies is immobilised in the analytical zone of the test-strip, whereas in a standard scheme of immunochromatography for the determination of specific antibodies the reagent for binding antibodies is conjugated with colloid gold and the antigen is immobilised in the analytical zone of the test-strip.

EFFECT: application of the claimed method for analysis makes it possible to increase the analysis sensitivity and/or reduce the antigen consumption.

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Description

The invention relates to immunology and medical diagnostics and is a method of immunochromatographic determination of specific antibodies. The presence in the blood serum of antibodies specific for the causative agent of a particular disease is an effective criterion for diagnosing the corresponding infectious disease with high confidence (Rose NR (ed.). Manual of Clinical Laboratory Immunology (4th ed.). Washington, DC: American Society for Microbiology, 1992.). 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.

Due to its 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 testing speed and performance 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 occurrence of specific reactions and the formation of immune complexes, which, due to the inclusion of a colored mark pa can be detected visually or by 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.

The sample, potentially containing specific antibodies, moves along the test strip under the action of capillary forces. At the same time, it first interacts with colored particles (colloidal gold or another marker), on the surface of which a component designed to bind to antibodies is adsorbed. Typically, 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 selected as such component, protein A from Staphylococcus aureus or protein G from Streptococcus spp. Then the front of the liquid overcomes the analytical (test) zone, which is a portion of the membrane with immobilized antigen (native or specially modified for effective sorption). 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-I test system of Vedalab (France) - see http://www.sanitamedikal.com/Assets/MD_220002_m3_TB_1408_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 a test sample passes through the adsorption zone of a 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. An unbound conjugate interacts with the reagent in the control zone of the test device, forming a colored strip, which indicates the proper conduct of the test ... Test procedure: ... fill the disposable pipette with serum or plasma and add 1 drop to the test device sample window. 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. To increase the sensitivity of the assay, the highest possible concentrations of the conjugate and receptor in the assay area should be used. However, an increase in the concentration of the colloidal conjugate is limited by the fact that the conjugate must completely diffuse through the immunochromatographic membranes during the analysis (~ 10 min); therefore, the use of conjugates with an optical density above 10 is problematic. The increase in the concentration of binding sites on the colloid surface is limited by the low sorption capacity of gold nanoparticles - usually a protein concentration of about 10 μg / ml is saturating (GHITESCU, L. and M. BENDAYAN. Immunolabeling efficiency of protein A-gold complexes. J. Histochem. Cytochem., 38, 1523-1530, 1990). The sorption capacity of the working membrane of the immunochromatographic test is several orders of magnitude higher than colloidal gold - up to 15 mg / ml (http://www.millipore.com/publications.nsf/a73664f9i981af8c852569b9005b4eee/348ee7096d9 3729b85256bf40066a40d500 / 00 FILE / $ FILE / FILE concentration of the receptor in the analytical zone in the standard method of immunochromatographic analysis for serodiagnostics is impractical for economic reasons, since the receptor is usually the most expensive components of the test system: recombinant proteins of bacteria or viruses. Therefore, we proposed a method for immunochromatographic analysis in which antigen or antigen molecules are adsorbed on the surface of colloidal gold (in this case, the antigen consumption is significantly reduced: from 300-600 ng to 100 ng per test), and the binding component is immobilized in the analytical zone of the test strip antibodies: anti-species antibodies, protein A from Staphylococcus uureus or protein G from Streptococcus spp. The listed reagents for binding antibodies are widely used in immunochemistry (in immunoassay, to highlight the purification of antibodies, etc.), therefore, there is a well-established mass production of these reagents and their cost is more than an order of magnitude lower than the cost of recombinant antigens, therefore a 10-fold increase in data concentration reagents does not lead to a significant increase in the cost of the test and at the same time can significantly increase the sensitivity of the analysis.

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

For the formation 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 membrane, and a GFB-applied sample membrane. R4, AP 045 adsorbing membrane; PT-R5 membrane was used for BSA deposition.

The following reagents were applied to the membranes:

1. Protein A from Staphylococcus aureus, manufactured by Imtek LLC (Russia).

2. The conjugate of colloidal gold with an average particle diameter of 30 nm and a recombinant antigen of 38 kDa M. tuberculosis (Rv0934), the company "Arista Biologicals Inc." (USA), cat. No. AGMTB-0220.

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

Protein A was used to form the analytical zone, and the NTM81 monoclonal antibodies against the 38 kD recombinant antigen M. tuberculosis were used as the control zone. 2 μl of Protein A solution (10.0 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 the strip. The conjugate of colloidal gold with antigen was applied in a dilution corresponding to D ₅₂₀ = 6.5, in a volume of 10 μl per 1 cm of the strip. For the application of reagents, an IsoFlow dispenser from Imagene Technology (USA) was used. After applying the reagents, the membranes were dried for at least 24 hours in a room with controlled temperature and humidity. Membrane sheets coated with immunoreagents were cut into individual test strips 3.5 mm wide.

For comparison, test strips were prepared according to the standard scheme using the same reagents and membranes, but with colloidal gold. Protein A was conjugated (10 μg / ml, the conjugate was applied to the membrane from an optical density of 7), and a recombinant 38 kDa M. tuberculosis protein was immobilized in the assay zone.

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 visually after 10 min (Fig. 2).

The sensitivity achieved using the proposed analysis method is an order of magnitude higher than the sensitivity achieved using the standard analysis method.

Fig. 1. The principle of immunochromatographic analysis for serodiagnosis. 1 - membrane adsorbing the sample; 2 - particles of colloidal gold; 3 - reagent for binding antibodies: anti-species antibodies, protein A, protein G or others; 4 - a plastic base; 5 - working membrane; 6 - final adsorbing membrane; 7 - antigen immobilized in the analytical zone; 8 - anti-species antibodies immobilized in the control zone.

Fig. 2. Testing of blood serum of patients with tuberculosis (3 samples): A - the standard method and B - the alternative method proposed by the applicants (the arrow indicates the position of the analytical zone).

Claims (1)

A method for immunochromatographic determination of specific antibodies, characterized in that a colloidal gold conjugate with an antigen is used for the analysis, and a reagent for binding common antibodies is immobilized in the assay area of the test strip, which can be used as an anti-species antibody, protein A from Staphylococcus aureus or protein G from Streptococcus spp.

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