RU2420740C1 - Method of immunoassay for analyte detection in sample - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: ferric (III) hexacyanferrate (II) hydrosol conjugated with antibodies (antigens) to a required analyte is used as a disperse phase. The presence of the analyte is detected by observing a bright blue strip on an analytical membrane. ^ EFFECT: faster immunoassay procedure as compared to conventional ones, higher contrast between an analytical zone and a white surface of the membrane. ^ 6 ex, 6 dwg

Description

Numerous examples of the use of immunochromatographic test systems for the detection of analytes are known (EP 0890103 B1; EP 1046913 A2; US 50968375238; US 5238652 ...). Similar examples are also described in the book "Lateral flow immunoassay" R.C. Wong, H.Y. Tse, eds., Humana Press NY, 2009. In these examples, gold nanoparticles (diameter 5-80 nm), stained or fluorescent polymer particles of submicron size (CA 2049919), nanoparticles of semiconductor materials are used as the dispersed phase for conjugation of antibodies (antigens) capable of luminescence (US 2006/0008921).

The closest analogue (prototype) of the present invention is described in patent EP 0890103 B1 "Quantitative immunochromatographic assay". In the device for carrying out the immunochromatographic analysis described in the prototype, it is assumed that the conjugate membrane contains artificially introduced microparticles, such as: colloidal metal particles, molecules of organic compounds, liposomes, or organic polymer latex particles. These particles are associated with a detecting antibody capable of binding to the analyte.

In the process of immunochromatographic analysis, a liquid sample containing an analyte interacts with microparticle antibodies, the complexes of analyte and microparticles formed by the capillary liquid flow are carried along the analytical membrane to a narrow analytical zone, in which there are capture antibodies that stop the microparticles bound to the analyte, forming a colored or luminescent complex.

This analysis method, in our opinion, has at least two drawbacks.

I. Colloidal metal particles, molecules of organic compounds, and especially liposomes or organic polymer latex particles, are expensive to manufacture and require complex methods for synthesizing the particles themselves, laborious chemical methods for bringing functional groups to the surface for subsequent binding to antibodies (antigens).

II. The migration rate along the analytical membrane of the analyte-microparticle complex and the rate of formation of the analytical signal are limited by convection and diffusion in the membrane [S. Qian, H.H. Bau. A mathematical model of lateral flow bioreactions applied to sandwich assays. / Analytical Biochemistry 2003, v. 333, pp. 89-98]. The diffusion coefficient in membranes with identical physical properties depends on the hydrodynamic radius of the microparticle complex with analyte and the viscosity of the eluting solvent. Typical analytical zone formation times when using colloidal gold microparticles with an average diameter of 35 nm, in membranes with different capillary flow rates (for example, Millipor HF120 and Millipor HF240), at room temperature, are from 20 to 45 minutes.

In addition, microparticles (especially particles of polymer latexes and liposomes) have the ability to aggregate, which causes a decrease in the rate of migration of the latter. This problem and methods of overcoming it are described in patent CA 2049919.

During immunochromatography, the front of the eluting solvent on the membrane is ahead of the propagation front of the microparticle complex with analyte, which leads to an uneven movement of the colored (luminescent) complexes and their aggregates along the analytical membrane, reducing the contrast between microparticles on the membrane and microparticles in the analytical zone, thereby lowering the sensitivity analysis. The dispersed phase we propose is a hydrosol of iron (III) hexacyanoferrate (II) devoid of the indicated disadvantages. The specified dispersed phase has the following advantages:

- The cost of the starting materials for its preparation and the final product itself are less expensive than dyed or fluorescent polymer latexes, metal (gold) colloids, liposomes.

- The process of synthesizing the hydrosol of hexacyanferrate (II) iron (III) is very simple, easy, and well reproducible.

- It has a well-developed surface and the ability to adsorb the binding of antibodies and antigens, both protein and lipopolysaccharide in nature.

- The movement of the microparticle complex with analyte along the immunochromatographic membrane occurs uniformly, simultaneously with the front of the eluting solvent. Typical immunochromatographic process time for Millipor HF120 membranes is 2 to 15 minutes. This is several times faster than for immunochromatography using other microparticles. Due to the uniform movement of the dispersed phase, the immunochromatographic membrane outside the analytical zone is practically not stained; high-contrast strips of bright blue color are formed in the analytical zone, making the analysis more sensitive.

The purpose of the invention is the detection of analytes in the test sample in a shorter time and with higher sensitivity.

Technical result: an acceleration of the immunochromatographic analysis procedure is provided, in comparison with gold sols, colored polymer latexes or quantum dots traditionally used as the dispersed phase, the contrast between the analytical zone and the white surface of the membrane is increased.

The claimed technical result is achieved due to the fact that the method of immunochromatographic analysis for the detection of analytes in a sample, including the creation of a multi-membrane composite consisting of a membrane for applying a sample, a conjugate membrane, an analytical membrane and a membrane for absorbing a sample containing a dry conjugate of a dispersed phase with an antibody (antigen) to one of the antigenic epitopes of the analyte, applying an immunoreagent (second antibody to another antigenic epitope of the analyte, or antigen) n and the analytical membrane in the form of a narrow zone, conducting an immunochemical reaction and separating the immunochemical complex simultaneously during chromatographic migration on the surface of the analytical membrane, is characterized in that the iron (III) hexacyanferrate (II) hydrosol is used as the dispersed phase for conjugation of the first antibody (antigen) .

The essence of the invention lies in the fact that in the immunochromatographic analysis used as a dispersed phase a hydrosol of hexacyanferrate (II) iron (III) conjugated with antibodies (antigens) to the desired analyte. An immunochromatographic analysis is carried out, the presence of an analyte is judged by the appearance of a strip painted in bright blue on the analytical membrane.

The method is as follows.

When carrying out the method, a conjugate of iron (III) hexacyanoferrate (II) hydrosol particles is obtained (GFCF; another name for this compound is “Prussian blue”) with antibodies to the analyte (antigen), or with an antigen to the analyte under investigation (antibodies), and the conjugate is applied to the conjugate membrane, followed by drying, a second antibody is applied to the antigenic epitope of the analyte, or antigen to the analytical membrane, in the form of a narrow strip in the analytical zone, a multi-membrane composite is formed, an immunochemical reaction is carried out on the surface the analytical membrane, followed by the determination of the analyte under study (antigen or antibodies) by the appearance of a colored band in the analytical zone. The analyte content in the test sample is proportional to the number of complexes of the conjugate of HCFC hydrosol particles with analyte that are captured in the analytical zone. The particles of the hydrophilic hydrophilic gel phase are bright blue, the color intensity of the band of the analytical zone is proportional to the amount of analyte in the sample.

EXAMPLES

Example 1. Obtaining conjugates of a hydrophosphate hydrophosphate gel with antibodies or with antigens

GCF hydrosol preparations are prepared by mixing solutions of ferric chloride and potassium ferric synergy in water.

To sensitize the surface of the particles of the hydrophobic hydrochloride with antibodies or antigens, an aqueous solution containing the immune component is added to the hydrosol preparation, the mixture is incubated on a magnetic stirrer for 30 minutes at room temperature and bovine serum albumin (BSA) is added to a final concentration of 0.1%. After 2 minutes of incubation under the same conditions, the mixture is centrifuged at 50,000-60000 xg for 30 minutes. The precipitate was suspended in a 0.1% BSA solution. The suspension is re-centrifuged under the same conditions. The precipitate was suspended in a 0.1% BSA solution. The absorption wavelength at the maximum and the optical density (OD) at the maximum are determined on a spectrophotometer. Using a solution of 5% sucrose, 0.25% BSA, hydrosol suspensions are prepared with a density (OD) of 3.0 at the maximum absorption and applied to the conjugate membrane. The specified membrane is dried for at least 24 hours in a room with a relative humidity of 20 to 30% and a temperature of plus 20 ° C - plus 25 ° C.

Example 2. Creating a multi-membrane composite

The Assembly of the composite (figure 1) is carried out at a relative humidity in the range from 20 to 30% at a temperature of from 20 to 25 ° C.

Solutions of antibodies to the antigenic epitopes of the analyte, or antigen (when determining antibodies as the analyte) are applied to the nitrocellulose membrane (3) in the area of the analytical zone in the form of a narrow strip (4). Anti-species polyclonal or monoclonal antibodies are applied to the control zone (5).

On the laminate substrate of the nitrocellulose membrane (6), a dried conjugate membrane (2) with a coated conjugate with OD = 3.0 and a membrane (1) for gluing a sample is glued. On the other hand, a membrane is glued onto the laminate substrate of the nitrocellulose membrane to absorb the sample (7). The assembled multi-membrane composite is cut into strips 5 mm wide and 60 mm long, which are placed in plastic mandrels (Fig. 2), the upper surface of which has a hole for depositing a sample (S) and a hole for reading the analysis results, indicated by the letters T and C.

Example 3. Determination of cholera exotoxin (XT)

A multi-membrane composite is used (Example 2), in the assay zone of which polyclonal rabbit anti-XT antibodies are applied, goat anti-rabbit antibodies are applied in the control zone, the conjugate substrate contains a conjugate of HCFC hydrosol particles with anti-XT antibodies. 120 μl of a solution containing 1% tween 20 and 1% BSA, or XT, in the same solution is added to the sample application hole.

The introduction of 120 μl of a solution of 1% tween 20 and 1% BSA into the hole for applying the sample leads to the appearance of an intense blue band in the control zone and its absence in the zone of the analytical band (Fig. 3A), which indicates the absence of nonspecific interaction of the conjugates of particles of the hydrophosphate gel containing antibodies to XT, with anticholinergic antibodies and serum components deposited in the analytical zone.

The introduction of XT into the test sample (source - cholera vaccine) leads to the appearance of two blue bands on the membrane: in the analytical (8) and control (9) zones (Fig. 3B). The signal intensity in the analytical zone increases with increasing analyte concentration in the sample and analysis time. The range of measured concentrations of XT from 2.0 to 500 μg / ml of the original solution for protein. A graph of the dependence of the intensity of the staining of the control zone on the concentration of XT in the sample is shown in Fig.4. Analysis time 2-15 minutes The signal intensity in the analytical zone is assessed visually or recorded using the Reflex video-digital analyzer.

If colored lines are not formed when the analyzed solutions are introduced into the hole for applying the sample (Fig. 2B), then such a test system is considered unsuitable, and its results are not taken into account.

Example 4. Determination in a sample of a blood serum titer of antibodies to the causative agent of salmonellosis

Use a multi-membrane composite (obtained according to example 2), in the analytical zone of which the preparation of Salmonella typhimurium lipopolysaccharide (LPS) was applied, monoclonal antibodies to the Salmonella group B O-antigen were applied to the control zone, the conjugate substrate contained a conjugate of HCFC hydrosol particles with sorbed LPS to typhimurium with OD = 3.0. To obtain the conjugate, LPS solutions with a concentration of 50 ng / ml and 200 ng / ml are used.

On a solution containing 1% tween 20 and 1% BSA, a series of successive working dilutions of the kS.typhimurium antiserum from 1:20 to 1:10 240 are prepared.

The introduction of 120 μl of a solution consisting of 1% tween 20 and 1% BSA into the sample opening leads to the appearance of a blue band in the control zone in the absence of a colored band in the analytical zone.

The introduction of AT into the above mixture to the pathogen of salmonellosis leads to the appearance of a blue band in the analytical zone, the intensity of which depends on the level of specific AT and reaction time. The range of measured titers AT to the causative agent of salmonellosis from 1:20 to 1: 5120. Figure 5 shows a graph of the intensity of the staining of the analytical zone from the dilution of antibodies to the causative agent of salmonellosis. Analysis time 1-15 minutes The signal intensity in the analytical zone is assessed visually or recorded using the Reflex video-digital analyzer.

Example 5. Determination in a sample of serum (plasma) of human blood of antibodies to the causative agent of tuberculosis

A multi-membrane composite is used (obtained according to example 2), in the analytical zone of which the preparation of a recombinant antigen, the glycoprotein complex Mycobacterium tuberculosis H37Rv, is applied, goat rabbit antibodies are applied to the control zone, the conjugate substrate contains a conjugate of HCFC hydrosol particles with antibodies of OD = 3.0 to the sum of human immunoglobulins of classes (IgG + IgM + IgA). On a solution containing 1% tween 20 and 1% BSA, a series of successive working dilutions of human serum (plasma) from 1: 2 to 1:10 are prepared, presumably containing antibodies to the causative agent of tuberculosis

The introduction of 120 μl of a solution consisting of 1% tween 20 and 1% BSA into the sample opening leads to the appearance of a blue band in the control zone in the absence of a colored band in the analytical zone (Fig. 2B), which indicates the absence of a sample of antibodies to the causative agent of tuberculosis. The introduction into the hole for the sample of 120 μl of a solution consisting of 1% tween 20 and 1% BSA and containing serum (plasma) from the blood of a person with tuberculosis leads to the appearance of two blue bands in the analytical zone (Fig. 2A), the intensity of which depends from the level of specific AT and reaction time. Analysis time 1-15 minutes The registration of results is carried out visually, or using the Reflekom device. If colored lines are not formed when the analyzed solutions are introduced into the hole for applying the sample (Fig. 2B), then such a test system is considered unsuitable, and its results are not taken into account.

Example 6. Determination of the time to reach the maximum analytical signal when using GFCF and colloidal gold as the dispersed phase of the hydrosol using the example of XT recording.

An example illustrates a reduction in analysis time for the same analyte, all else being equal.

Received a multi-membrane composite with GTsFZh (as described in example 2) and a multi-membrane composite with colloidal gold.

To obtain conjugates of colloidal gold particles with antibodies to XT, an aqueous solution containing anticholeogenic antibodies is added to the colloidal gold preparation, the mixture is incubated on a magnetic stirrer for 10 min at room temperature, and BSA is added to a final concentration of 0.1%. After 1 min of incubation under the same conditions, the mixture is centrifuged at 50,000 × g for 30 minutes. The suspension is washed twice with a solution containing 10% sucrose and 0.25% BSA, each time precipitating particles by centrifugation under the same conditions. The precipitate was suspended in 0.02 M phosphate buffer, pH 7.9, containing 10% sucrose and 0.25% BSA. The absorption wavelength at the maximum and the OD value at the maximum are determined on a spectrophotometer. Using the same buffer solution, a suspension of a gold sol is prepared with an OD equal to 3.0 at the maximum absorption, and applied to the conjugate membrane. The membrane is dried as described in example 1. The assembly of the multi-membrane composite is carried out as described in example 2.

120 μl of XT solution prepared in 0.1 M phosphate buffered saline pH 7.9 containing 0.5% BSA and 0.4% Tween 20 is added to the hole for applying a sample of a mandrel containing a conjugate of colloidal gold particles with anticholeogenic antibodies.

XT is determined as described in Example 3. The signal intensity in the analytical zone is recorded using a Reflexom video-digital analyzer. A graph of the dependence of the signal intensity in the analytical zone on the analysis time is shown in Fig.6. When using an HFC liquid hydrosol as the dispersed phase, the maximum signal in the zone of the analytical band appears 5 min after the sample was introduced. When using gold sol conjugates, a longer time is required to achieve the maximum analytical response (more than 40 min).

All of the examples described above are characterized by the rapid movement of particles of the HCFC hydrosol conjugate along the nitrocellulose membrane and the appearance of colored bands in the analytical and control zones, which distinguishes immunochromatographic tests using HCFC hydrosol preparations from immunochromatographic tests based on gold sols, for which the analysis time is 15-40 min At high analyte concentrations, a signal visible to the eye appeared 1-2 minutes after sample administration. The particles of the hydrophilic hydrocyclone hydrocyclone sensitized by an antibody or antigen passed through the analytical membrane with a smooth front, practically giving no “smudges” or “sagging”. The background of the analytical membrane was from white to light blue, the formed lines in the analytical and control zones were distinguished by good contrast.

An increase in the contrast between the analytical zone and the white surface of the membrane is ensured by more complete removal of the staining hydrosol of iron (III) hexacyanferrate (II) and, as a result, improvement of visual or instrumental recording of the analysis results.

Claims (1)

A method for conducting an immunochromatographic analysis for detecting analytes in a sample, comprising creating a multi-membrane composite consisting of a membrane for applying a sample, a conjugate membrane, an analytical membrane and a membrane for absorbing a sample containing a dry conjugate of a dispersed phase with an antibody (antigen) to one of the antigenic epitopes of the analyte , applying an immunoreagent (second antibody to another antigenic epitope of the analyte or antigen) on the analytical membrane in the form of a narrow zone, conducting immunoha nical immunochemical reaction and the separation of the complex at the same time during a chromatographic analysis of migration on the surface of the membrane, characterized in that as the dispersed phase for conjugating the first antibody (antigen) is used hydrosol geksatsianferrata (II) iron (III).

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