RU232546U1 - Immunochromatographic test system for multiplex analysis - Google Patents

Abstract

The utility model relates to devices for immunochromatographic determination of infectious agents and toxins of bacterial and plant origin and can be used in healthcare, biotechnology and immunology. The test system is designed for rapid (up to 20 minutes) detection in samples from environmental objects (water, air, soil, etc.) of at least 24 types of biopathogens: viruses, microorganisms, toxins. The test system consists of immunochromatographic test strips, 4 test strips combined into a cartridge. The utility model is distinguished by the fact that the analytical (test) and control zones are made in the form of microdots with a diameter of 0.3-0.7 mm. The test strip includes 2 control and at least 6 test (analytical) microzones. Moreover, each test microzone contains antibodies for detecting one biopathogen, and the cartridge is designed to detect at least 6 biopathogens on a separate strip and at least 24 biopathogens in one sample using 4 test strips of one cartridge.

The test system is intended for use in immunochromatographic analysis devices with time-resolved luminescence.

Description

The utility model relates to devices for immunochromatographic determination of pathogens of infectious diseases and toxins of bacterial and plant origin and can be used in healthcare, biotechnology and immunology.

The claimed immunochromatographic test system is intended for in vitro detection of antigens of viruses, microorganisms and toxins in samples taken from objects of the external environment, including in samples of aerosols transferred to the aqueous phase. The utility model is intended for use by health care professionals in sanitary and epidemiological laboratories, including for the prevention of threats associated with acts of bioterrorism.

The object of patenting is the design of a multiplex immunochromatographic test system designed for the simultaneous detection of up to 24 types of biopathogens in a sample. Variants of the design of the test system also allow it to be used for the simultaneous analysis of 4 samples for the presence of 6 biopathogens of one of the listed groups.

Multiplex test systems for the immunochromatographic determination of analytes are known (Zhang X. et al. // Journal of Agricultural and Food Chemistry. - 2017. - Vol. 65. N. 36. - P. 8063-807; Kong D. et al. //Nanoscale. - 2016. - Vol. 8. N. 9. - P. 5245-5253) in the form of cartridges of several test strips, including a holder (a plastic frame with windows for introducing a sample and reading an optical signal). The test strip contains a substrate for laminating porous materials, on which a filter pad for applying the test sample, a pad for applying the conjugate, a porous membrane and an absorption pad are located sequentially along the length and with an overlap. The pad for applying the conjugate is impregnated with a conjugate of colloidal gold particles. The number of analytes detected on one test strip is determined by the number of analytical zones located across the flow of liquid (test sample). Analytical zones are biospecific binding reagents (antigens, antibodies, DNA probes, etc.) applied as a narrow strip 0.5-1 mm wide at a distance of at least 2 mm from each other. As a rule, the number of analytical zones on one test strip does not exceed 4.

There are known designs of cartridges in which nanoparticles with europium ion complexones are used as a developing reagent in test strips instead of colloidal gold conjugates (Chen K. et al. // Int J Environ Res Public Health. - 2021. - Vol. 18. N. 9. - P. 4574. doi: 10.3390/ijerph18094574; Valanne A. et al. // J. Clin. Virol. - 2005. - Vol. 33. - P. 217-223.; Jaakohuhta S. et al. // Int. J. Food Microbiol. - 2007. - Vol. 114. - P. 288-294). In this case, the signal from the test strip is recorded in the luminescence time-resolved mode using specialized devices - fluorimeters with time resolution.

The time-resolved mode provides increased sensitivity due to the ability to detect lower concentrations of fluorescent marker than with photometric detection of the signal from colloidal gold particles.

Known analogs of test systems use cartridges for multiplex analysis on one test strip from one to three or four analytes due to the arrangement of analytical zones in the form of stripes across the immunochromatographic flow. The cartridge design includes a housing, which is a plastic frame with a window for introducing a sample and windows for viewing the results, inside which a substrate, a pad for applying a liquid test sample and conjugate, a filtration pad, a porous chromatographic membrane and an absorption pad are placed.

The pad for applying the sample and conjugate is made of cellulose or glass microfiber, or polyester microfiber with a pore size of 20-25 μm and an absorption capacity of at least 55 mg/cm2 ^.

The filter pad is made of porous polysulfone material with a layer thickness of 400-500 µm and a pore volume of 55-57 µl/ ^cm2 .

The porous membrane is made of nitrocellulose, or polyethersulfone, or nylon with a layer thickness of 80-150 microns.

The absorption pad is made of porous cotton fluff or glass cellulose fiber with a layer thickness of 700-900 microns.

The lamination substrate is made of polystyrene or acrylic adhesive material with a layer thickness of 230-270 microns.

Biospecific immunoreagents are applied to the surface of the porous membrane in the form of analytical and control transverse stripes.

The choice of materials and their characteristics is not limited to the specified parameters, but is determined as a result of optimization for specific types of desired analytes.

The specified technical elements taken together are necessary and sufficient for the correct functioning of the immunochromatographic cartridge.

At the same time, the specified design has limitations both in terms of the level of multiplexity and in terms of the level of sensitivity when detecting extremely low concentrations of analytes. Thus, the application of analytical (test) zones in the form of transverse stripes significantly limits the possibility of placing a larger number of analytical zones. In addition, the inclusion of a developing reagent (a conjugate of nanoparticles with a biospecific component, such as antibodies) in the composition of test strips does not allow completely eliminating the influence of the conjugate outside the analytical microzones, which limits the sensitivity of the analysis, since about 4% of the mixture of sample and conjugate is distributed on the membrane surface and creates a luminescent background level that limits the determination of low concentrations of analytes.

There are known designs of cartridges (test systems) consisting of one test strip in a case, in which the multiplexity is increased and is not limited to 3-4 analytes on one test strip. To increase the multiplexity, the analytical zones are made in the form of microdots with a diameter of 0.5-1 mm. Colloidal gold nanoparticles are used as a developing (detecting) system, which are located in the starting zone of the test strip on a separate absorbent pad. Such designs consisting of one test strip and a pad with colloidal gold nanoparticles in the starting zone have limitations in multiplexity and sensitivity of the analysis.

Increasing the multiplexity of the analysis on such test strips due to a denser arrangement of microzones requires the use of developing reagents in the form of a mixture for each analyte or in the form of a universal multipurpose reagent in a higher concentration than is necessary when analyzing only one analyte, which is inevitably accompanied by a decrease in the sensitivity of the analysis due to an increase in the background level from the unbound reagent. The design of these test systems excludes the possibility of washing the test strips to completely remove the developing reagents.

There are known designs of cartridges (test cassettes) in which the increase in multiplexity is achieved by combining several test strips into one design for contact with one sample of 4 test strips at once. In this case, the design of the test cassette is designed for immersion of the starting end of the test strip into the sample. In the known prototype (utility model RU 208858, IPC G01N 33/558 for determining antibiotics), the increase in the multiplexity of the analysis is limited to 4 test and one control lines on the test strip, which ensures the detection of no more than 16 analytes on 4 test strips of the test cassette.

The sensitivity of analyte detection in the prototype is limited by photometric or visual detection of the signal from colloidal gold nanoparticles, which is hundreds of times lower than when detecting a signal from long-luminescent nanoparticles.

Unlike the prototype discussed above using a cartridge of several test strips, where the analytical (test) and control zones are made in the form of transverse strips, the design of the test system proposed by us in the form of a cartridge of 4 test strips with applied point microzones solves the problem of increasing the multiplexity and sensitivity of the analysis simultaneously. At the same time, the proposed design reduces the consumption of immunoreagents for the formation of test and control microzones by 10 times.

Structurally, the test system (cartridge) is made in the form of 4 multiplex immunochromatographic test strips with microdots of biospecific reagents (immunoglobulins) applied to the membrane surface. A long-luminescent marker in the form of nanoparticles with europium ion complexons is used as a developing reagent. In this case, the nanoparticles contain a biospecific component covalently bound on the surface in the form of a specific immunoglobulin to a biopathogen or a mixture of specific immunoglobulins to several biopathogens or streptavidin.

Fig. 1 shows the appearance of the test system (cartridge). The cartridge includes 4 test strips (A, B, C, D) with two control microzones (microdots) 1, 2 and 6 analytical (3-8) on each test strip.

Analytical microzones contain specific mouse or rabbit immunoglobulins.

Control microzones contain anti-species antibodies against mouse and rabbit immunoglobulins.

Microzones (1, 2 Fig. 1) contain anti-species antibodies for binding mouse and (or) rabbit specific antibodies. Microzones 3-8 are intended for detection of the desired analytes. Each of the microzones 3-8 includes antibodies for detection of only one type of biopathogen. The location of specific antibodies is determined by its number.

The Bactoplex test strip (Fig. 1A) is designed to detect pathogens of plague (3), anthrax (4), tularemia (5), brucellosis (6), legionellosis (7), glanders and melioidosis (8).

The test strip "viroplex1" (Fig. 1B) is designed to detect the causative agents of Crimean-Congo hemorrhagic fever CCHF (3), dengue fever (4), West Nile fever (WNF) (5), RIVT Valley fever (RVF) (6), yellow fever YF (7), and Japanese encephalitis (JE) (8).

The test strip "viroplex2" (Fig. 1 C) is designed to detect pathogens of encephalitis and encephalomyelitis: eastern (VsEL) (3), Venezuelan (VEL) (4), western (WEL) (5), tick-borne (TB) (6), as well as vaccinia virus (7) and Sars-cov2 (8).

The test strip "Toxiplex" (Fig. 1D) is designed to detect botulinum toxins of types A (3), B (4), as well as a polyvalent test strip for the determination of botulinum toxins of types A, B, E, C, F (5), ricin (6), cholera toxin (7), staphylococcal enterotoxin type B (SEB) (8).

The design of the test strips provides for the possibility of including other analytical microzones in order to detect other types of biopathogens, in addition to those listed above, as well as excluding individual microzones when there is no relevance in determining the corresponding analytes. The same applies to specific antibodies in the composition of the developing reagents.

The cartridge design provides for its installation in the wells of the microplate and immersion of the free end of the test strips in the sample volume in the well of the microplate with subsequent incubation of test trips with the sample and reagents in the wells of the 96-well microplate. The test strips have only one catching pad and do not contain pads for the sample and conjugate. This design of the test strip allows for incubation of the sample in the wells of the microplate with a subsequent washing stage (incubation of the test strips with the analysis buffer) to remove luminescent nanoparticles that have not bound in the microzones. The washing stage is possible only after transferring the cartridge from the wells of the microplate with samples and developing reagents to the wells with the analysis buffer. The absence of a pad for the sample and conjugate in the test strip allows for the complete removal of luminescent nanoparticles from the free volume of the membrane composite within 5 minutes, thereby increasing the signal/background ratio by more than 10 times and increasing the threshold level for detecting biopathogens several times compared to the traditional design containing a pad for the conjugate and sample introduction.

For multiplex detection of one or more of the 24 types of biopathogens using a test system (cartridge), various versions of the developing reagent can be used:

- in the form of a mixture of at least 6 types of monospecific nanoparticles for the detection of at least 6 types of biopathogens of bacterial nature using the test strips "bactoplex", of viral nature - using the test strips "viroplex1", "viroplex2" and of toxin nature - using the test strips "toxiplex";

- in the form of nanoparticles, which are a conjugate of several (at least 6 types) antibodies, bound on the surface with a luminescent nanoparticle. Each test strip of the cartridge contacts only one type of nanoparticle, specific to bacteria, viruses or toxins;

- in the form of mixtures of biotinylated antibodies and one universal developing reagent - nanoparticles conjugated with biotin. All the listed types of specific immunoreagents ensure the detection of at least 24 types of biopathogens by the test system.

Table 1 presents the immunological materials used for the preparation of analytical and control microzones of the cartridge test strips for the detection of toxins and the corresponding detecting immunoreagents (biotinylated antibodies and nanoparticles).

Table 2 presents immunoreagents for the preparation of test strips for the detection of microorganisms, as well as detecting immunoreagents (nanoparticles and biotinylated antibodies).

Table 3 presents immunoreagents for the preparation of test strips for the detection of viruses and detecting immunoreagents (nanoparticles and biotinylated antibodies).

Immunoreagents are applied to test strips using a contactless printing nanoplotter manufactured by IMMUNOSKRIN LLC. Antibody concentration is 2 mg/ml, quantity in a microzone is 20 nl, spot diameter is 0.5-0.6 mm. The distance between individual microzones does not exceed 1.5-2 mm.

The proposed technical solution of the test system in the form of a cartridge of 4 test strips, each of which contains at least 8 (test and control) microzones, is designed to detect on each test strip one group of at least 6 types of biopathogens.

The number of microzones on the test strip can be increased to 20-25 for a 5x20 mm scanning area, since to resolve luminescent signals from each microzone, it is necessary that the density of microzones does not exceed one microzone per 4 ^mm2 when using a commercially available automated immunochromatographic analyzer (utility model RU No. 230227).

The utility model expands the functional capabilities of immunochromatographic analysis by increasing multiplexity, which meets the criterion of novelty. The consumption of immunoreagents for the formation of test and control microzones (instead of lines) is reduced by 10 times compared to the prototype.

The utility model can be used in medical clinical diagnostics, in laboratory practice of the sanitary and epidemiological service, in law enforcement agencies to ensure biological protection of troops and the population. Consequently, the utility model meets the criterion of industrial applicability.

Below is an example of setting up an analysis of several samples using the proposed utility model. To demonstrate the operability of the test system, samples were prepared that included biopathogen imitators. Each sample contained one imitator for each test strip of the cartridge. A total of 6 samples were prepared. Sample No. 1 contains biopathogen imitators that should be identified based on the signal level in zone No. 3 of each test strip. In sample No. 2, biopathogens should be identified based on the signal level in zone No. 4 of each test strip, etc.

Fig. 2 graphically shows the results of scanning the luminescent signal from the surface of the membrane composite of test strips after 10 minutes of contact of the sample with a mixture of the analysis buffer and a developing reagent in the form of nanoparticles with a diameter of 50 nm, including antibodies to 6 types of biopathogens and a subsequent 5-minute incubation of the test strips of the cartridge with the analysis buffer to remove free luminescent nanoparticles from the membrane. Luminescent nanoparticles and their conjugates with biospecific antibodies were obtained according to TU 20.59.52-199-260-05031637-2017 using the technology described in the laboratory regulations (No. LR - 05031637-150-2017). The content of biopathogens in the sample and the results of the analysis (signal levels) during detection on the IFI-05 pulsed photoluminescence indicator device (reg. No. RZN 2022/16369 dated 21.02.2022) depending on the type of developing reagents used are shown in Tables 4, 5, 6. Inactivated toxins (anatoxins), sucrose-acetone antigens (SAA) of viruses and bacterial preparations were used as imitators.

The results for determining the same biopathogens using a mixture of 6 types of long-lasting luminescent nanoparticles, each of which is a conjugate with only one type of antibody, in an analysis on a separate test strip of the test system are presented in Table 5.

Table 6 presents the results of biopathogen detection using universal long-term luminescent nanoparticles conjugated with streptavidin and a mixture of biotinylated antibodies to biopathogens. Each test strip of the cartridge was incubated with a mixture of 6 types of biotinylated antibodies and then with nanoparticles with streptavidin.

All three types of the used developing reagents (data in Tables 4, 5, 6) include long-luminescent nanoparticles with a diameter of 50±7 nm. The obtained signal/background ratios for all three types of developing reagents give close ratios. At a concentration of nanoparticles in the sample (10 ⁹ pcs. / 200 μl), the maximum absolute signal levels in the control and analytical (test) microzones are observed when using a mixture of 6 types of long-luminescent nanoparticles as a developing reagent, and the minimum - when using biotinylated antibodies and long-luminescent nanoparticles covalently bound to streptavidin. For all 3 variants of developing reagents, the differences in absolute signal levels do not exceed a twofold value.

In the test system, particles with a diameter larger than 50 nm can be used as part of the developing reagent.

With an increase in the diameter of the nanoparticles used (from 50 to 300 nm), their concentration in the analysis is reduced proportionally to the surface area of the nanoparticles. In this case, the signal level increases proportionally to the volume of the nanoparticles.

The analysis with samples and reagents was carried out using the wells of a 96-well microplate. The cartridge test strips were incubated with the samples and reagents by immersing their free (start) end into the wells of the microplate. The length of the free (start) end was from 5 to 15 mm. After the stage of washing the membrane with the analysis buffer, the background luminescence level decreases by more than 10 times (data not shown) and the I _c /I _f ratio in the microzones, accordingly, increases proportionally to the decrease in the background level.

Tables 4, 5, 6 present the results of the analysis obtained according to the time cyclogram, including a 10-minute incubation cycle of the cartridge with samples and developing reagents and a subsequent 10-minute cycle of washing the cartridge test strips in the analysis buffer.

The results presented in Tables 4, 5, 6 confirm the possibility of using the utility model to increase the multiplexity of the analysis of biopathogens using various developing reagents and to increase sensitivity due to the possibility of effective washing of the membrane composite by successive immersion of the starting end of the test strips into wells with a sample, reagents and washing buffer.

The utility model (test system) is a consumable and is used in the operation of an automated immunochromatographic analysis system (patent for utility model RU No. 230227 “Automated immunochromatographic analyzer”).

Claims (3)

1. An immunochromatographic test system for multiplex analysis of biopathogens, including immunochromatographic test strips combined into a cartridge, characterized in that the test and control microzones of the test strip are made in the form of microdots, the test strips are combined into a cartridge, which includes at least 4 test strips, for detecting at least 6 types of biopathogens on each of the test strips. 2. The test system according to item 1, characterized in that the starting end of the test strips, which is in contact with the sample, reagents and wash buffer, protrudes from the cartridge body by 5-15 mm. 3. The test system according to item 1, characterized in that the test and control microzones of the test strips are made of biological materials in the form of microdots with a diameter of 0.3-0.7 mm.