RU218447U1 - Multiparametric test system for the simultaneous determination of antigens of SARS-CoV-2 viruses, influenza type A and B by immunochromatography - Google Patents

Abstract

The utility model is designed to determine the nucleocapsid antigens of SARS-CoV-2, influenza type A, influenza type B in respiratory samples, namely in human nasopharyngeal and oropharyngeal swabs. Can be used as a tool for laboratory diagnosis of coronavirus infection COVID-19 and influenza. The device is a test strip - a complex of membranes on a plastic substrate with pre-applied reagents, including a control zone and a number of analytical zones with monoclonal antibodies specific to the SARS-CoV-2 virus and influenza A and B viruses, a membrane for conjugates with a colored marker . The membrane for conjugates with a colored marker was pretreated with a buffer solution with the addition of a composition of organic polymers of polyethylene glycol (PEG) 1%, polyvinylpyrrolidone (PVP) 0.25% and Pluronic f68 poloxamer 0.5%. The device provides uniform washing out of the colored reagent during analysis, which ensures uniform distribution of conjugates across the membrane, minimizing non-specific binding and resolubilization without loss of reactivity. Thus, high values of the analytical parameters of the test system are achieved. 2 ill.

Description

The utility model relates to the determination of nucleocapsid antigens of SARS-CoV-2 viruses, influenza A and B in a clinical sample in a short time by immunochromatographic analysis.

COVID-19 infections and influenza caused by these viruses are acute respiratory illnesses with similar symptoms and transmission patterns. Fever, myalgia, headache, sore throat, cough, or runny nose are common in mild infections (WHO Coronavirus Infection (COVID-19): COVID-19 and Influenza 2021). However, complications up to a lethal outcome are possible. For example, acute respiratory distress syndrome (ARDS) with the development of respiratory failure (Pormohammad, A., et al., 2021; Shchelkanov, M., et al., 2015). The risk of complications depends on the timely initiation of etiotropic therapy. Thus, a tool is needed for accurate differential diagnosis of these infections (Cuadrado-Payán, E., et al., 2020). Rapid detection of a pathogen will allow choosing the right treatment strategy, minimizing the consequences of the disease, and updating information on the epidemiological situation in time.

Modern laboratory diagnostics of COVID-19 and influenza includes the polymerase chain reaction (PCR) method (Yatsyshina, S., et al., 2016; Kuznetsova, N.A., et al., 2020; Zhdanov, K., et al. ., 2020; Tsang, N.N.Y., et al., 2021) and various immunoassays (Di Domenico, M., A. De Rosa, and M. Boccellino, 2021; Steininger, C., et al., 2020). PCR diagnostics and most immunochemical methods combine a low detection limit and high reproducibility of results. However, their application requires expensive equipment and qualified personnel, and the determination itself takes a long time.

An alternative method that does not have these disadvantages is immunochromatographic analysis. You can evaluate the result of the study visually in 15 minutes, which corresponds to the modern trend in medical diagnostics - analysis "at the point of care" ("Point-of-care testing").

Thus, thanks to the use of immunochromatographic test systems, it becomes possible to detect the disease and interrupt the chains of transmission of infection at an early stage through targeted isolation and surveillance of the majority of infected and close contacts. Simultaneous detection of SARS-CoV-2 antigens and influenza A and B viruses minimizes the number of samples taken, reduces analysis time and cost, and makes the test a convenient tool for emergency departments or outreach.

However, the development of such systems is a complex and knowledge-intensive process. The creation of a stable and sensitive immunochromatographic test system is preceded by careful selection and individual optimization of a number of parameters. One of the key elements of the test strip is the conjugate membrane. Here, the formation of the first immunospecific complexes with a colored marker occurs. The task of the membrane is to maintain the stability of the conjugate in the dried form, to facilitate its effective leaching to the working area, to control the rate of movement of the liquid and reagents.

A frequent problem in the development of a multiparametric test is the violation of the kinetics of the movement of the liquid front during the transition from the membrane under the conjugate to the working membrane due to the large amounts of heterogeneous and prone to aggregation conjugates of the stained marker and antibodies. Additional pretreatment (Millipore E. M. D. Rapid lateral flow test strips: considerations for product development, 2013) of the membrane with the proposed buffer eliminates these difficulties. Thus, a uniform distribution of the conjugate over the working membrane is ensured without phantom bands and spots that make it difficult to detect the result.

Membrane pretreatment buffer components include organic polymers polyethylene glycol (PEG) 1%, polyvinylpyrrolidone (PVP) 0.25%, and Pluronic f68 poloxamer 0.5%. They minimize non-specific binding, promote uniform and slower sample movement, thereby increasing the specific interaction time and shifting the detection limit to lower concentrations. This option is the easiest to implement the test and does not require the introduction of new signal amplification systems. Taking into account the specifics of the claimed utility model, publications on the development of multiparametric test systems, where the membrane is pretreated for conjugate, are considered below.

1) a test system for the simultaneous detection of cane sugar mosaic viruses, described in the publication of Thangavelu R. M. and co-authors "Ultrasensitive nano-gold labeled, duplex lateral flow immunochromatographic assay for early detection of sugarcane mosaic viruses" early detection of sugar cane mosaic viruses”): Scientific reports, 2022, v.12. p. 1-14. DOI: 10.1038/s41598-022-07950-6.

2) a test system for the simultaneous detection of atrazine and acetochlor pesticides, described in the publication of Ruan X. et al. "Nanomaterial-enhanced 3D-printed sensor platform for simultaneous detection of atrazine and acetochlor" simultaneous determination of atrazine and acetochlor"): Biosensors and Bioelectronics, 2021, v.184. p. 113238. DOI: 10.1016/j.bios.2021.113238.

The test system described in the publication by Thangavelu R. M. and co-authors is a membrane composite, which includes:

1) membrane for the sample;

2) membrane for conjugate, pretreated with sodium phosphate buffer pH 7.4 with the addition of 2% BSA, 2% PVP, 1% sucrose and 0.25% Tween-20;

3) nitrocellulose membrane with immobilized mAbs against sugarcane mosaic and striated mosaic viruses in the respective test areas and rabbit anti-species antibodies in the control area;

4) final adsorbing membrane for absorbing sample components after the reaction.

For the detection of viral antigens, cysteamine-coated mAb and NPSC conjugates corresponding to the virus are used. The conjugates of each mAb type are applied to a separate membrane and dried. When assembling the test strip, both membranes are placed overlapping.

At the end of the analysis, "golden" amplification is used, which consists in the reduction of gold salts. This leads to the formation of large particles and, consequently, a greater intensity of staining on the membrane.

The analysis is carried out as follows:

1) Plant extract is applied to the test strip, incubation occurs.

2) The result of the analysis is fixed visually.

The result of the analysis is interpreted as follows:

1) If one colored line appears on the working membrane in the control zone, the result is considered negative. The sample does not contain antigens of sugar cane mosaic and striated mosaic viruses;

2) If two or three lines are stained on the working membrane, one of which is the control one, the result is considered positive. The sample does not contain antigens of sugar cane mosaic and striated mosaic viruses.

The technical result of the utility model is to improve the conditions for the flow of immunochemical reactions on the membrane of the test system with the achievement of the minimum limit of detection of nucleocapsid antigens of SARS-CoV-2 viruses, influenza A and B.

The specified technical result is achieved by the fact that before applying the mAb conjugates against SARS-CoV-2 viruses, influenza type A and B with a colored marker, the membrane is pre-treated with a buffer solution with the addition of a composition of organic polymers of polyethylene glycol (PEG) 1%, polyvinylpyrrolidone (PVP) 0.25% and poloxamer Pluronic f68 0.5%.

The buffer solution with the addition of a composition of organic polymers of polyethylene glycol (PEG) 1%, polyvinylpyrrolidone (PVP) 0.25% and poloxamer Pluronic f68 0.5% is dissolved in the applied conjugate, mixed with it and dried. During the analysis, after the introduction of the sample, it contributes to the uniform washing out of the colored reagent from the membrane. Uniform distribution of conjugates across the membrane minimizes non-specific binding and ensures their resolubilization without loss of reactivity. Thus, due to the pretreatment of the membrane for the conjugate with a buffer solution with the addition of a composition of organic polymers of polyethylene glycol (PEG) 1%, polyvinylpyrrolidone (PVP) 0.25%, and Pluronic f68 poloxamer 0.5%, high values of the analytical parameters of the test system are achieved.

The test system described by Ruan X. and co-authors is a bifunctional membrane composite, which includes:

1) an expanded membrane for a triangular sample;

2) two membranes for the conjugate, one of which was pretreated with a solution of 2% BSA + 2% sucrose, the other with a solution of 2% BSA + 2% sucrose + 0.3% Tween-20;

3) two nitrocellulose working membranes: on one of them, the atrazine-BSA complex was applied in the test zone, on the other, the acetochlor-BSA complex. Goat anti-species antibodies were adsorbed in the control zone;

4) two end adsorbing membranes for absorbing sample components after the reaction.

Antibody conjugates with platinum-palladium nanoparticles (Pd@Pt NPs) are used for pesticide detection.

Pesticides are determined by competitive analysis.

The analysis is carried out as follows:

1) 200 µl of the analyzed sample is applied to the membrane under the sample, incubation takes place for 10 minutes.

2) The reacted sections of the working membrane are cut out with a holder knife and lowered into the reaction cells filled with thionine acetate and hydrogen peroxide. Platinum-palladium nanoparticles as part of the immune complex catalyze the redox reaction (ORR) between these reagents. The OVR process is controlled voltammetrically using a potentiostat.

The content of pesticides in the sample is quantified.

To achieve high analytical parameters of test systems, the authors of publications present a whole range of solutions aimed at signal amplification. The main approach is to use a non-classical version of the marker and/or instrumental registration of the result. However, this method of detection requires the use of high-tech equipment and additional work with reagents, which makes point-of-care analysis impossible and is not suitable for self-testing. Such systems are also difficult in full-scale production: the complexity of assembling systems limits the possibilities of process automation, and the introduction of signal amplification systems will increase the cost of materials and then affect the cost of analysis.

It should be noted that, in these test systems, the membranes for the conjugate, although they are subjected to special treatment, but the conjugates of different mAbs are applied to separate membranes. Thus, pretreatment of the membrane with a solution is not a single complex solution to the problem of placing conjugates on one membrane, effectively washing out the marker on the working area of the test strip, and increasing the sensitivity of the system - this is just one of the elements of a number of optimizations.

Moreover, BSA, which is part of the pretreatment solution, does not contribute to uniform washing out of the components, and when dried, it can potentially reduce the chemical stability of the system, oxidize, deaminate, exhibit conformational instability and promote aggregation (Horn, J., Mahler, H.-C. and Friess, W. (2020), Drying for Stabilization of Protein Formulations, In Drying Technologies for Biotechnology and Pharmaceutical Applications (eds S. Ohtake, K.-i. Izutsu and D. Lechuga-Ballesteros), https://doi.org /10.1002/9783527802104.ch4).

The technical objective of the claimed utility model is to improve the conditions for the flow of immunochemical reactions on the membrane of the test system to achieve the minimum limit of detection of nucleocapsid antigens of SARS-CoV-2 viruses, influenza type A and B. The technical result of the utility model is to use a special composition for pretreatment of the membrane for conjugate . When applied, the composition dissolves in the applied conjugate, mixes with it and contributes to uniform washing out of the colored reagent during the analysis. As a result, the selected formulation maintains uniform distribution of the conjugates across the membrane, minimizing non-specific binding and resolubilization without loss of reactivity. Thus, the found optimum provides high values of the analytical parameters of the test system.

The specified technical result is achieved by the fact that:

conjugates of mAbs against the SARS-CoV-2 virus, influenza A and B viruses with a colored marker are applied to the membrane for the conjugate;

Before applying the conjugates to the membrane, it is pre-treated with a special solution of PEG 1%, PVP 0.25% and Pluronic f68 0.5%.

To carry out the analysis, as shown in Fig. 1:

1) test strips are prepared, on which all the necessary immunoreagents are applied;

2) respiratory specimens (human nasopharyngeal and oropharyngeal swabs) are diluted with working sodium phosphate buffer in a volume of 0.7 ml;

3) 3 drops (90 µl) of the resulting solution are applied to the absorbent membrane of the test strip and incubated for 10 minutes;

4) Based on the formation of colored lines, the result is interpreted.

The described analysis includes the following processes:

If the analyzed sample contains nucleocapsid antigens of SARS-CoV-2 viruses and/or influenza type A and/or influenza type B, they react with the corresponding specific mAbs labeled with a colored marker. Immune complexes "antigen-mAb-stained marker" are formed, which continue to move with the fluid flow. In the corresponding analytical zone of the test strip, interaction with immobilized specific mAbs against SARS-CoV-2 and/or influenza A and/or B virus occurs with the formation of stained immune complexes “immobilized mAb–sample antigen–mAb–stained marker”. An increase in the amount of such immune complexes leads to the formation of a visible colored line in the corresponding antigen of the analytical zone of the test strip. In the control zone of the test strip, a specific stained immune complex of anti-species antibodies and mAbs in the conjugate is formed regardless of the presence of an antigen in the sample.

The interpretation of the results of the analysis is carried out in accordance with the scheme presented in fig. 1: when one colored line appears in the control zone (7), it is concluded that there are no nucleocapsid antigens of SARS-CoV-2 viruses, influenza A and B in the test sample. When two or more colored lines appear, one of which is the control line (7), a conclusion is made about the presence of nucleocapsid antigens of SARS-CoV-2 viruses and/or influenza type A and/or influenza type B in the test sample.

The effectiveness of this approach is confirmed by the following example below:

Using the claimed device, analysis of model samples is carried out with the content of nucleocapsid antigens of SARS-CoV-2 viruses, influenza A and B in the concentration range of 0-0.6 ng/ml. Samples were obtained by serial dilutions of recombinant virus antigens in sodium phosphate buffer.

3 drops (90 μl) of the resulting solution are added to the surface of the membrane under the sample. After 10 minutes, the analysis results are documented and their subsequent digital processing.

Figure 2 shows a graph of the obtained dependence of the staining intensity of the analytical zone (the result of digital image processing of test strips) on the concentration of nucleocapsid antigens of SARS-CoV-2 viruses, influenza A and B in the sample. From the obtained data, it can be seen that the created test is capable of identifying these antigens with a detection limit of 0.01 ng/ml, 0.06 ng/ml, 0.15 ng/ml, respectively (the beginning of an increase in staining intensity, i.e. the appearance of a line in analytical zone), which exceeds the performance of the test system assembled without using the proposed composition.

Brief description of the drawings

On fig. 1 shows the scheme of the test strip: 1 - absorbent membrane, 2 - working sodium phosphate buffer for diluting the sample, 3 - pretreated membrane for conjugate, 4 - analytical zone of the working membrane with sorbed mAbs against type B influenza, 5 - analytical zone of the working membrane with adsorbed mAbs against type A influenza, 6 – analytical zone of the working membrane with adsorbed mAbs against SARS-CoV-2, 7 – control zone of the working membrane with adsorbed anti-species antibodies, 8 – adsorbing membrane.

On fig. Figure 2 shows the calibration curves for the immunochromatographic determination of nucleocapsid antigens of SARS-CoV-2 viruses, influenza A and B in model samples in the concentration range of 0–0.6 ng/ml: 9 – determination of antigens of the SARS-CoV-2 virus in a system with membrane pretreatment for conjugate, 10 - detection of antigens of the SARS-CoV-2 virus in the system without membrane pretreatment for conjugate, 11 - determination of antigens of influenza A virus in the system with membrane pretreatment for conjugate, 12 - determination of influenza type A antigens in the system without membrane pretreatment for conjugate, 13 - determination of antigens of type B influenza virus in the system with pretreatment of the membrane for conjugate, 14 - determination of antigens of influenza type B in the system without pretreatment of the membrane for conjugate.

Claims (1)

A device for the determination of nucleocapsid antigens of SARS-CoV-2 viruses, influenza types A and B by immunochromatography, which is a test strip - a complex of membranes on a plastic substrate with pre-applied reagents, including a control zone and a number of analytical zones with monoclonal antibodies specific to the virus SARS-CoV-2 and influenza A and B viruses, a membrane for conjugates with a colored marker, characterized in that the membrane for conjugates with a colored marker is pretreated with a buffer solution with the addition of a composition of organic polymers of polyethylene glycol (PEG) 1%, polyvinylpyrrolidone (PVP) 0 .25% and Pluronic f68 poloxamer 0.5%.

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