RU2789545C1 - Method for highly sensitive competitive immunochromatographic analysis - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to analytical biochemistry and food biotechnology, in particular to a method for competitive immunochromatographic analysis, which is based on a combination of two immune interactions of antibodies labeled with a stained label. According to the present invention, instead of an antigenic preparation applied to the membrane, it is assumed to use a protein-bound structural analog of an antigen capable of binding to antibodies, but with worse affinity than the antigen itself. Due to this, even small concentrations of antigen in the sample become sufficient to prevent the formation of stained complexes on the membrane. Since the proposed replacement reduces the intensity of staining in the absence of antigen in the sample, making it difficult to compare the analysis results for different concentrations of antigen, the replacement is accompanied by the use of an additional reagent: a conjugate of a marker particle with an antibody-binding reagent.

EFFECT: invention makes it possible to reliably detect lower concentrations of low molecular weight antigen.

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Description

The invention relates to analytical biochemistry and food biotechnology, representing a method for competitive immunochromatographic analysis.

Immunochromatographic analysis includes methods for detecting various compounds that use the binding of these compounds to specific antibodies and the formation of a complex of immunoreagents and a detectable (usually colored) label, which is detected by the appearance on the test strip, by means of antigen-antibody reactions on the immunochromatographic membrane (test strip). colored band [Quesada-Gonzalez D., Merkoci A. Nanoparticle-based lateral flow biosensors. BIOSENSORS & BIOELECTRONICS, 2015, v. 73, pp. 47-63]. The demand for immunochromatographic analysis is determined by the simplicity of its implementation - the contact of the test strip with a liquid sample or extract and, if necessary, with additional solutions is sufficient to initiate the movement of liquid along the membrane under the action of capillary forces and initiate all interactions necessary for the formation of detectable immune complexes, thereby analysis does not require additional devices and labor-intensive actions of the operator. The result of immunochromatographic analysis is the presence or absence of a colored binding zone in a certain area of the test strip, due to the binding of immunoreagents in this area, including their complexes with a label. Thanks to this, the evaluation of the test results can be carried out visually, and the entire analysis can be carried out directly at the sampling site without transporting them to the laboratory.

If it is necessary to determine low molecular weight compounds, which include pesticides, antibiotics, mycotoxins, phycotoxins and other toxic contaminants of agricultural products and food and feed products produced from it, a single-site competitive immunochromatographic analysis is used, which is due to the ability of small-sized antigen molecules to bind to only one antibody molecule [Posthuma-Trumpie G.A., Korf J., van Amerongen A. Lateral flow (immuno) assay: its strengths, weaknesses, opportunities and threats. A literature survey. ANALYTICAL AND BIOANALYTICAL CHEMISTRY, 2009, v. 393(2), pp. 569-582]. Competitive immunochromatographic analysis is based on a combination of two immune interactions of antibodies labeled with a colored label. Antibodies are first contacted with a test sample that potentially contains an antigen, and during this contact the antibodies specifically bind to the antigen. Then the sample, mixed with the antibody preparation, moves through the pores of the test strip membrane under the action of capillary forces. An antigenic preparation associated with a protein is applied in advance to a certain zone of this membrane. Usually, the antigen itself is used for this, into which a reactive group is introduced (or an unmodified antigen, if its molecule already contains such a group), which covalently binds to a protein that provides sorption immobilization of the resulting complex at the site of application to the membrane. As a result of these two interactions, on the membrane of the test strip in the zone of application of the antigen bound to the protein, when the concentration of the antigen in the sample is below the threshold, a complex registered by staining is formed (carrier protein - antigen derivative - antibodies - marker particle). If the antigen concentration in the sample is equal to or higher than the threshold, the complex is not formed in an amount sufficient for registration by staining. Thus, based on the result of staining registration, the unknown concentration of antigen in the sample is compared with the threshold concentration. If the concentration of the antigen in the sample is below the threshold, staining is observed; if it is equal or higher, it is absent. It is important that comparison with the threshold concentration of the analysis allows us to draw practically significant conclusions about the properties of the test sample, for example, whether it exceeds the permissible concentration of a toxic contaminant - pesticide, antibiotic, etc. However, the threshold concentration of a competitive immunochromatographic analysis is a certain value that depends on the structure of the antigen and antibodies, the binding constants of their interaction. If the assay detects lower concentrations of antigen than required for practical purposes, it can still be used effectively after diluting the test sample. If the threshold concentration of immunochromatographic analysis is higher than the controlled level of the detected antigen, it is necessary to improve the analytical method [Ngom B., Guo, Y., Wang X. Et. al. Development and application of lateral flow test strip technology for detection of infectious agents and chemical contaminants: a review. ANALYTICAL AND BIOANALYTICAL CHEMISTRY, 2010, v. 397(3), pp. 1113-1135].

As you can see, increasing the sensitivity of immunochromatographic analysis is a highly demanded task. Various approaches are proposed to solve it.

One of the directions for improving immunochromatographic tests is associated with the replacement of the used marker. Instead of gold nanoparticles, which are used in most immunochromatographic systems, it is proposed to use other antibody-binding particles that are detected at lower concentrations. Thus, successful developments are known in which fluorescent or magnetic particles are used, which are recorded in extremely low quantities and with a low background signal of the membrane and components of the tested samples [Urusov A.E., Zherdev A.V., Dzantiev B.B. Towards lateral flow quantitative assays: Detection approaches. BIOSENSORS, 2019, v. 9 (3), article 89]. However, the implementation of such an immunochromatographic analysis requires additional recording devices, which increases the cost of determination and leads to the loss of the main advantage of immunochromatography as a non-instrumental analytical method implemented directly at the sampling point.

There are also developments in which nanoparticles of a different chemical composition and, accordingly, with other absorption spectra are used as an alternative label [Goryacheva I.Yu., Lenain P., De Saeger S. Nanosized labels for rapid immunotests. TRENDS IN ANALYTICAL CHEMISTRY, 2013, v. 46, pp. 30-43]. However, a change in the surface properties of a nanoparticle affects the efficiency of immobilization of antibodies on its surface and the degree to which they retain their antigen-binding properties. Thus, the advantages of a more intensely colored label can only be realized after a set of additional studies to select the conditions for immobilization of antibodies on its surface.

In addition, as an alternative label in immunochromatographic analysis, it is proposed to use nanozymes - nanoparticles that catalyze the transformation of a substrate into a detectable colored product. At the same time, due to the transformation of a large number of substrate molecules, the amplification of the optical signal is indeed provided [Nguyen Q.H., Kim M.I. Nanomaterial-mediated paper-based biosensors for colorimetric pathogen detection. TRENDS IN ANALYTICAL CHEMISTRY, 2020, v. 132, article 116038]. However, the assembly of the test system becomes more complicated. Instead of a single test strip with pre-applied and dried reagents, there is a combination of test strip and substrate solution, the properties of which can change over time, which affects the recorded intensity of staining.

The effect on the threshold concentration of competitive immunochromatographic analysis of the composition of complexes of nanoparticle-labels with antibodies was noted [Zvereva, E.A., Byzova N.A., Sveshnikov P.G. et al. Cut-off on demand: adjustment of the threshold level of an immunochromatographic assay for chloramphenicol. ANALYTICAL METHODS, 2015, v. 7 (15), pp. 6378-6384]. Indeed, a decrease in the ratio of antibody:label in the complex from many tens and hundreds to units shifts the threshold concentration to lower values, since a smaller number of antigen molecules contained in the sample is required to block the antigen-binding sites of antibodies. However, with a smaller amount of antibodies in a complex with a nanoparticle, the efficiency of binding to the immobilized conjugate of the antigen derivative with a protein carrier on the membrane also decreases, which manifests itself as a decrease in the staining intensity and less reliable registration of the results obtained.

The literature also has proposals for changing the properties of the conjugate of the antigenic drug with protein that affect the binding of antibodies [Anfossi L., Baggiani C., Giovannoli C., Giraudi G. Laderel Flow Immunossays for Aflatoxin B and G and For Aflatoxin M1. Chapter 215 in Aflatoxins - Recent Advances and Future Prospects Intechopen, 2013. Pp. 315-339]. So, it is proposed to change the length of the “bridge” -(CH ₂ ) _n -the area of the antigenic molecule and the carrier protein, the location of the modification point on the antigen molecule. In some cases, changes also lead to a more sensitive definition of free antigen. However, the success of such a modification is not guaranteed, and the achieved winnings can vary greatly. In addition, a change in the conjugation methodology of a modified antigen with protein requires additional optimization of synthesis conditions and may be associated with a decrease in the output of the target product.

Thus, to solve the problem of increasing the sensitivity of immunochromatographic analysis, different methods can be used, however, such changes may be associated with an increase in the complexity of the analysis or require additional studies when choosing the conditions for obtaining the necessary new reagents.

At the same time, for such popular classes of controlled low molecular weight compounds as pesticides, antibiotics, non-protein toxins, a significant variety of structurally similar molecules is known, with varying degrees of efficiency recognized by antibodies - commercial chemicals of the same class with several modified or replaced atoms, products of partial destruction of antigens. The characterization of antibodies used to determine a particular antigen includes such a parameter as cross-reactivity, which reflects the results of a comparison of binding to antibodies of a given antigen derivative and the original molecule. [Sotnikov D.V., Zherdev A.V., Zvereva E.A. et al. Changing cross-reactivity for different immunoassays using the same antibodies: Theoretical description and experimental confirmation. APPLIED SCIENCES, 2021, v. 11 (14), article 6581].

Given the availability of such reagents, the present patent proposes to use a protein-bound derivative of a structural analog of the antigen, capable of binding to antibodies, but with a worse affinity than the antigen itself, instead of the antigenic preparation deposited on the membrane. Due to this, even small concentrations of antigen in the sample become sufficient to prevent the formation of colored complexes on the membrane. Since the proposed replacement reduces the intensity of staining in the absence of antigen in the sample, making it difficult to compare the results of analysis for different concentrations of antigen, the replacement is accompanied by the use of an additional reagent - a complex of stained particles with an antibody-binding protein that moves along the test strip through its pores after the antigen- antibody. The binding of this additional reagent provides a high intensity of staining in the absence of antigen in the sample and the ability to reliably compare the results of the analysis for different concentrations of antigen.

According to the mathematical model of competitive immunochromatography described in the literature [Sotnikov D.V., Zherdev A.V., Byzova N.A., et al. Mathematical modeling of the immunochromatographic test-systems in competitive format: Analytical and numerical approaches.. BIOCHEMICAL ENGINEERING JOURNAL. (2020), v. 164, article 107763] the signal in the analytical zone is determined by the concentration of the stained complex, which depends on the parameters of the system as follows:

Where

A - antigen in the sample;

P - antigen-binding centers of antibodies on marker particles (marker conjugate);

R - modified antigen (antigen-protein) in the analytical zone for binding the conjugate of the colloidal marker;

AP - antigen-antibody complex on a colloidal marker particle;

PR - a complex of specific antibodies on particles of a colloidal marker and a modified antigen in the analytical zone;

ka1 - kinetic constant of the association of the reaction of labeled antibodies with the antigen;

ka2 - kinetic constant of the association of the reaction of labeled antibodies with a modified antigen in the analytical zone;

t is the time of analysis from the beginning of the reaction of the antigen with the colloidal conjugate;

T is the difference between t and the reaction time in the analytical zone.

As can be seen from the equation, the concentration of the colored complex in the analytical zone is in a power-law dependence on the ratio of the association constants of the first and second antigen-antibody reactions. Quantitatively, the sensitivity of the analysis is related to the value of the abscissa of the inflection point of the calibration dependence (IC50). The lower the concentration of competitor corresponding to the IC50, the higher the sensitivity. The immune interaction constants that determine the rates of reactions (1) and (2), ka1 and ka2, respectively, affect the concentration of the colored PR complex formed in the analytical zone in a fundamentally opposite way: [PR] increases with a decrease in ka1 and an increase in ka2. Calculations show that an increase in the value of ka1 leads to a shift in IC50 towards lower values of the analyte, illustrating the well-known requirement to use as high affinity antibodies as possible in the analysis. In this case, a change in ka1 has little effect on the value of the maximum concentration [PR]max achievable in the absence of the analyte. In contrast, a decrease in the kinetic association constant of the marker conjugate with the receptor in the assay zone (ka2) shifts the IC50 towards lower analyte values, but the maximum concentration [PR] decreases significantly, i.e., the sensitivity increases, but the color intensity decreases. It follows from the model that it is more efficient to use high concentrations of a low-affinity receptor in the assay zone than low concentrations of a high-affinity receptor.

Accordingly, the subject of the invention is a method of competitive immunochromatographic analysis, in which a less affine structural analogue of the antigen with respect to antibodies is used as an antigen preparation associated with a protein to increase sensitivity, and after interactions between the antibody complex and the marker particle, the conjugate moves along the membrane of the test strip a marker particle with an antibody-binding reagent to enhance the color intensity.

The proposed combination of substitutions in the configuration of the immunochromatographic test system makes it possible to detect lower concentrations of the antigen while maintaining the required characteristics of the analysis - methodological simplicity, registration of membrane staining as a result of testing, no need for instrumentation, maintaining a high intensity of staining.

The technical result of the patent is to increase the sensitivity of the analysis, that is, to enable reliable detection of lower concentrations of the antigen.

The feasibility of the proposed idea is demonstrated by the following example:

Example 1

Immunochromatographic analysis of the antibiotic sufachlorpyridazine

Analysis of sufachlorpyridazine is carried out using antibodies specific to this compound, and sulfadimethoxine is used to obtain a complex of an antigenic preparation with a protein carrier (cross-reactivity with the antibodies used, according to enzyme immunoassay, -17%).

Immunochromatographic test strips were prepared using a CNPC nitrocellulose membrane with a pore size of 15 µm (CNPC-SS15), a GFB-R4 sample membrane, a PT-R7 glass fiber membrane, and an AP045 adsorption membrane (Advanced Microdevices, India).

Spherical gold nanoparticles (average diameter 30 nm) were obtained by reducing chloroauric acid with sodium citrate according to the Frens method, and polyclonal rabbit antibodies to sufachloropyridazine and streptococcal protein G were immobilized on them by adsorption at pH 9.0, based on the flocculation curve.

The analytical zone of the test strip was formed by applying a conjugate of sulfadimethoxine with bovine serum albumin (0.5 mg/ml), the control zone was formed with anti-species antibodies (immunoglobulins of sheep antiserum against rabbit IgG (0.15 mg/ml). For comparison, we also used the immobilization of the conjugate of sufachlorpyridazine with bovine serum albumin (0.5 mg/ml), a complex of gold nanoparticles with antibodies to sufachlorpyridazine was applied to a glass fiber membrane.

After applying the immunoreagents, the nitrocellulose membrane was dried for 24 hours at room temperature and 5 hours at 37°C. The dried and glued membrane complex was cut into test strips 3.5 mm wide using an automatic guillotine cutter.

Immunochromatographic analysis (control experiments with immobilized sulfonylamide conjugate with bovine serum albumin): 100 µl of the test sample (solutions of sufachlorpyridazine of various concentrations) were added to the well of the microplate and the test strip was immersed in a vertical position. After 10 min, the result of the analysis was monitored.

Immunochromatographic analysis (suggested version with immobilized conjugate of sulfadimethoxine with bovine serum albumin): 100 μl of the test sample (solutions of sufachlorpyridazine of various concentrations) were added to the well of the microplate and the test strip was immersed in a vertical position. After 10 minutes, the test strip was transferred to another well containing 100 μl of the conjugate of gold nanoparticles and streptococcal protein G. The 10-minute incubation was repeated and the result of the analysis was monitored.

The results of the analysis for two options - the control and the proposed one, are shown in fig. 1 reflect the shift in the threshold concentration from 10 to 3.3 ng/ml of sufachlorpyridazine in the sample, i.e. confirm the claimed improvement in the sensitivity of the competitive immunochromatographic assay.

Claims (1)

A method for highly sensitive competitive immunochromatographic analysis of low molecular weight antigens, including: 1) binding of antigen molecules potentially contained in the test sample with antibodies to the antigen being determined in combination with a colored marker particle, 2) subsequent movement of the sample along the membrane of the test strip and binding of the complex of antibodies and marker particles by means of unreacted antigen-binding sites of antibodies with a protein-bound antigenic preparation deposited on the test strip membrane in the analytical zone of the test strip, characterized in that the protein-bound antigenic preparation uses a structural analogue of the antigen that is less affine with respect to antibodies , and after the interaction of the antibody complex and the marker particle, the conjugate of the marker particle with the antibody-binding reagent is added to the test strip membrane, which ensures high staining intensity in the analytical zone of the test strip in the absence of antigen in the sample.

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