RU2733260C1 - Method for increasing sensitivity of immunochromatographic test systems with silver lactate and hydroquinone - Google Patents

Abstract

FIELD: immunochromatographic analysis.

SUBSTANCE: invention relates to immunochromatographic analysis. Method for increasing the sensitivity of immunochromatographic test systems with silver lactate and hydroquinone involves preliminary preparation of two membranes – nitrocellulose impregnated with 0.075 % silver lactate solution, and a membrane treated with 3 % hydroquinone solution prepared in 0.5 M citrate buffer with pH 4.0; assembling the ready multi-membrane composite of the immunochromatographic test system by gluing on the nitrocellulose membrane with the immobilized conjugate, the sample membranes and the absorbent membranes; testing the prepared immunochromatographic test system by adding 100 mcl of sample to the sample membrane; 10 minutes after introduction of sample on detectable zone of test system membrane impregnated with 3 % hydroquinone solution; applying on superimposed membrane 30 ml of 0.01 M Tris-HCl buffer with 1 M NaCl with 0.1 % Tween-20, pH 7.5, 5 minutes later membrane is removed with hydroquinone to detect antigen in test region.

EFFECT: invention provides higher sensitivity of immunochromatographic test systems for detecting both infectious and non-infectious agents, at least four times.

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Description

This invention relates to the field of immunochromatographic analysis and can be used to increase the sensitivity of the developed immunochromatographic test systems.

The immunochromatographic test system is a multimembrane composite consisting of a nitrocellulose membrane, a membrane with an immobilized conjugate of mono- or polyclonal antibodies to a detectable antigen with a marker, a membrane for introducing a sample, and an adsorbing membrane. Colloidal gold nanoparticles are most often used as a marker in immunochromatography.

There are various techniques for amplifying the optical signal generated by colloidal gold nanoparticles in test systems.

A technique based on the use of enzymes is known from the prior art [Parolo, C. Enhanced lateral flow immunoassay using gold nanoparticles loaded with enzymes / C. Parolo, A. de la Escosura-Muñiz, A. Merkoçi // Biosensors and Bioelectronics. - 2013. - V. 40. - P. 412-416.]. An immunochromatographic test system is proposed, in which gold nanoparticles are conjugated with antibodies and peroxidase. After the immunochromatographic analysis, the test strip is washed to remove unbound reagents and immersed in a solution of peroxidase substrate (tetramethylbenzidine), which gives a bright blue insoluble oxidation product. Enhanced staining allowed the detection limit to be reduced from 5 ng cm ^-3 to 200 pg cm ^-3 .

Drygin Yu.F. and co-authors proposed another method for enhancing the analytical signal in immunochromatography, which consists in using silver nanoparticles with a diameter of 20 nm, aggregating on the surface of colloidal gold nanoparticles (NCG) and in
10 times increasing the intensity of staining [Drygin, Yu.F. Highly sensitive immunochromatographic express method for determining the infection of plants with the tobacco mosaic virus / Yu.F. Drygin, A.N. Blintsov et al. // Biochemistry. - T. 74. - No. 9. - 2009. - S. 986-993].

The silver enhancement technique is based on the physical manifestation response used in photography. In addition to the reducing agent, the "physical" developer contains ions or metal complexes. From such solutions, metals are deposited onto the centers of the latent image. In histochemical silvering, NchKZ act as such centers. Nanoparticles catalyze the reduction of silver ions from salt to metallic silver. The shell of metallic silver around the gold particle, in turn, accelerates the reduction and the reaction becomes autocatalytic. For the first time this technique was used for histochemical analysis in 1981. The author used hydroquinone as a reducing agent, and from silver salts he preferred lactate [Dykman, L.А. Gold nanoparticles. Synthesis, properties, biomedical application [Text] / L.А. Dykman, V.A. Bogatyrev, S.Yu. Shchegolev, N.G. Khlebtsov // M: Science. - 2008. - S. 195-196].

Closest to the invention is an immunochromatographic test system for the detection of protein CagA H. pylori [Bogacheva, N. In. Immunochromatographic test system for detecting pathogenic strains of Helicobacter pylori / N.V. Bogacheva, I. V. Darmov, D.N. Smirnova // Patent for invention RU 2642588 C1, IPC G01N33 / 543, G01N33 / 577, application No. 2017108557 dated 03/14/2017, published: 01/25/2018 in Bul. Number 3]. This test system is a multimembrane composite consisting of a membrane for a sample, an adsorbing membrane, a nitrocellulose membrane, on the surface of which a membrane impregnated with a conjugate of monoclonal antibodies, clone HP-387 (Bialexa, Russia), with nanoparticles of well gold with an average diameter 30 nm, and applied in the transverse direction monoclonal antibodies, clone HP-1811 (Bialexa, Russia), in the test and anti-species goat antibodies against mouse Ig (Bialexa, Russia) - in the control zones.

The authors propose a method for increasing the sensitivity of immunochromatographic test systems using silver lactate and hydroquinone using the example of a test system for the detection of H. pylori CagA protein.

The technical result of the invention is the development of a method for increasing the sensitivity of immunochromatographic test systems intended for the detection of both infectious and non-infectious agents, not less than fourfold.

The technical result is achieved by working out the options for applying silver lactate to the components of the multimembrane composite, choosing the concentration of silver lactate, assessing the effect of hydroquinone on the amplification of the optical signal in the analytical zone of the test system, followed by determining the sensitivity threshold of the experimental sample of the immunochromatographic test system reinforced by the above immunochemical components during testing with museum cultures of H. pylori and AGHPY-0100 antigen.

The method of increasing the sensitivity of immunochromatographic test systems consists in preliminary preparation of two membranes - nitrocellulose, impregnated with 0.075% silver lactate solution, and a membrane treated with 3% hydroquinone solution, prepared in 0.5 M citrate buffer with pH 4.0; assembling a ready-made multimembrane composite of an immunochromatographic test system with immunochemical components applied to the membranes; testing the finished immunochromatographic test system by adding 100 μl of the sample to the membrane for the sample; the imposition of a membrane impregnated with 3% hydroquinone 10 minutes after introducing the sample onto the detectable zone of the test system; application of 30 ml of acceleration buffer to the superimposed membrane, exposure for 5 min, removal of the membrane with hydroquinone and determination of the detectable antigen in the test zone.

The resulting increase in the sensitivity of the immunochromatographic test system was fourfold confirmed by examples of testing the developed immunochromatographic test system for detecting the H. pylori CagA protein , both when testing with a culture of a microorganism, and when testing the AGHPY-0100 antigen, which contains intracellular H proteins pylori associated with cagA (120 kd) and vacA (87 kd) genes and urease.

We used monoclonal antibodies to the H. pylori cagA protein, clones HP-1811 and HP-387 (Bialexa, Russia); goat anti-species antibodies against mouse IgG (Bialexa, Russia); hydrochloric acid (GOST 3118-77); silver lactate (Sigma Aldrich, USA); hydroquinone (Russia); antigen AGHPY-0100 ("Arista Biologicals"); sodium citrate (Reakhim, Russia); citric acid (Reakhim, Russia); tris (hydroxymethyl) aminomethane C4H11NO3 (Sigma, USA); tris hydrochloride C4H12ClNO3 (Panreac, Spain); Tween-20 (AppliChem, Germany).

The multimembrane complex includes membranes from MDI, India: nitrocellulose membranes TYPE-CNPF-SN12-L2-P25 8μ and 10μ; membranes for deposition of samples TYPE-GFB-R4 (0.35); glass fiber membranes for the PT-R5 conjugate; membranes for absorbent TYPE-AP 045.

All solutions were prepared in deionized water. To obtain deionized water, we used the UF Arium 611 UF system (Sartorius, Germany).

The stages of developing a method for increasing the sensitivity of immunochromatographic test systems with silver lactate and hybroquinone will be considered on the example of improving the immunochromatographic test system for detecting H. Immunochromatographic test system for detecting pathogenic strains of Helicobacter pylori / N.V. Bogacheva, I. V. Darmov, D.N. Smirnova // Patent for invention RU 2642588 C1, IPC G01N33 / 543, G01N33 / 577, application No. 2017108557 dated 03/14/2017, published: 01/25/2018 in Bul. Number 3].

Obtaining an experimental sample of an immunochromatographic test system for the detection of H. pylori CagA protein .

A conjugate of 30 nm colloidal gold nanoparticles with MkAT HP-387 was prepared according to the procedure specified in the patent for invention RU 2642588 C1.

The conjugate solution was impregnated onto the conjugate membranes at a density of 30 μl per cm ² and dried in air at room temperature for 24 hours.

For the formation of the test zone, MkAT HP-1811 was used, for the formation of the control zone, goat anti-species antibodies against mouse immunoglobulins at a concentration of 10 mg × cm ^-3 . The test systems were dried at room temperature for a day.

The gluing of the test systems was carried out sequentially: a membrane with an immobilized conjugate, a membrane for a sample, and a membrane for an adsorbent were glued onto a nitrocellulose membrane. Then the obtained multimembrane composite was cut with a cutter (Rahmenlos® Katze Geschenk Shirt, USA) into separate strips 3.5 ÷ 4.0 mm wide.

The developed experimental sample of the immunochromatographic test system is illustrated in FIG. 1, which shows the composition of the multimembrane composite and the main immunochemical components of the test system:

1 - membrane for applying the TYPE-GFB-R4 sample (0.35);

2 - conjugate of NchKZ with MkAT HP-387 applied to the PT-R5 membrane;

3 - MKAT HP-1811 at a concentration of 12 mg × cm ^-3 ;

4 - TYPE-CNPF-SN12-L2-P25 10μ nitrocellulose membrane;

5 - anti-species goat antibodies against mouse Ig at a concentration of 10 mg × cm ^-3 ;

6 - TYPE-AP 045 adsorption membrane.

Strengthening the test system with silver lactate and hydroquinone

A 0.075% silver lactate solution was applied to the nitrocellulose (working) membrane of the immunochromatographic test system. Left to dry in the dark for a day. The resulting membrane was used for the manufacture of immunochromatographic test systems.

A 3% hydroquinone solution prepared in 0.5 M citrate buffer with pH 4.0 was applied to a PT-R5 glass fiber membrane. The membrane was dried at room temperature in a dark place.

When testing the immunochromatographic test system, after applying the test material to the membrane for the sample, we waited 10 min until the signal appeared in the control and, if the sample contained H. pylori CagA protein, in the analytical zone, and then applied over the nitrocellulose membrane, in the detection area, the membrane made of PT-R5 fiberglass impregnated with 3% hydroquinone solution. Over the membranes, 30 μL of acceleration buffer (0.01 M Tris-HCl buffer with 1 M NaCl with
0.1% Tween-20, pH 7.5). After 10 min, the membrane with hydroquinone was removed and the result was taken into account.

The invention is illustrated by examples of comparative assessment of the sensitivity of unmodified and modified, enhanced with silver lactact and hydroquinone, variants of the test system with a cag + H. pylori strain and AGHPY-0100 antigen (Arista Biologicals, USA).

Example 1. Testing of unmodified and modified variants of the immunochromatographic test system with the H. pylori strain .

The cag + H. pylori strain was used to assess the sensitivity . This strain was isolated from biological materials of persons with a history of gastritis and gastric ulcer (the contents of periodontal pockets and biopsies of the gastric mucosa). The belonging of cultures to H. pylori was confirmed by bacteriological and biochemical methods, and to the cag + strain of H. pylori - by PCR using commercial test systems (Helikopol VA, Helikopol CA, Helikopol IA, Helikopol BA ( Russia), "DNA-technologies", Russia) and by the method of immunochromatography using a commercial test system ("NovaMed", Israel).

For testing, we used a culture of H. pylori diluted with an acceleration buffer starting from a concentration of 10 × 10 ⁹ with a "step of two" to a concentration of 0.31 × 10 ⁹ mc × cm ^-3 . The runaway buffer was used as a control.

On the membrane for a sample of unmodified and modified test systems, 100 μL of the test sample at a certain concentration was applied. In the case of the modified variant, after applying the culture, after 10-15 minutes, a PT-R5 membrane impregnated with a 3% hydroquinone solution was applied over the test and control zones of the test systems.

The results were determined visually for 20-25 minutes. In the presence of the H. pylori CagA protein in the sample, when it is applied in a volume of 100 μl onto the sample support, the antigen interacts with the conjugate of mAb with NchKZ, forming a colored antigen-antibody immunocomplex, which moves under the action of capillary forces along the nitrocellulose membrane, crosses the test zone and interacts with specific mAbs immobilized on the indicated test zone - the test zone is colored pink. Further, the conjugate unbound in the test zone continues to move along the chromatographic membrane and interacts with anti-species rabbit antibodies against mouse immunoglobulins immobilized in the control zone. As a result, the control zone also turns pink, which is the internal control of the test. If the analysis is carried out correctly, it should always appear, regardless of the presence of antigen in the test sample.

The results of testing the developed variants of test systems with H. pylori culture shown in Fig. 2, indicate a fourfold decrease in the detection threshold - from 2.5 × 10 ⁹ m.c. × cm ^-3 to 6.25 × 10 ⁸ m.c. × cm ^-3 .

Example 2. Testing of the immunochromatographic test system with the antigenic preparation AGHPY-0100 ("Arista Biologicals", USA)

The antigenic preparation AGHPY-0100 (Arista Biologicals, USA) contains H. pylori intracellular proteins associated with the cagA (120 kd) and vacA (87 kd) genes and urease.

The study of the sensitivity of the immunochromatographic test system with the antigenic preparation AGHPY-0100 was carried out in the same way, using unmodified and modified with silver lactate versions of the test systems. The antigen was diluted with run-off buffer from 0.5 to 0.062 mg × cm ^-3 and applied 100 μl to the sample membrane. The result was evaluated after 20-25 minutes.

The results of the analysis of the developed variants of the test systems with the antigenic preparation, presented in Fig. 3, indicate a fourfold decrease in the detection threshold, from 0.5 mg × cm ^-3 to 0.125 mg × cm ^-3 .

Claims (1)

A method for increasing the sensitivity of immunochromatographic test systems with silver lactate and hydroquinone, which consists in the fact that two membranes are preliminarily prepared - a nitrocellulose impregnated with a 0.075% silver lactate solution and a membrane treated with a 3% hydroquinone solution prepared in a 0.5 M citrate buffer with pH 4.0; collect the finished multimembrane composite of the immunochromatographic test system by sticking on the nitrocellulose membrane with the immobilized conjugate, the membrane for the sample and the membrane for the absorbent; test the finished immunochromatographic test system by adding 100 μl of the sample to the membrane for the sample; impose a membrane soaked in 3% hydroquinone solution on the detectable zone of the test system 10 minutes after introducing the sample; 30 ml of 0.01 M Tris-HCl buffer with 1 M NaCl with 0.1% Tween-20, pH 7.5 are applied to the superimposed membrane, after 5 minutes the membrane with hydroquinone is removed and the detected antigen is determined in the test zone.

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