RU77687U1 - LAYING IMMUNOCHROMATOGRAPHIC INDICATOR ELEMENTS - Google Patents

Abstract

The utility model relates to stackings for a specific indication of microorganisms and toxins. The laying of immunochromatographic indicator elements is intended for sampling from the external environment and identifying causative agents of especially dangerous infectious diseases (OOIs): anthrax, plague, tularemia, glanders, as well as type A botulinum toxin by immunochromatography. The technical result of the claimed utility model is to shorten the analysis procedure, incl. in the process of studying washouts from hard surfaces, and the exclusion of "false positive results" of analyzes. The claimed technical result is achieved due to the fact that the laying of immunochromatographic indicator elements, containing inside the hermetically sealed enclosure: kits for sampling, kits for analysis of the sample, pencils, scissors, tweezers, a glass recorder, a bag for sterilization of waste, latex gloves, nameplate, shipping container , characterized in that it contains a hair brush in the sampling kit, contains at least one indicator immunochromatographic element, which is a set of membranes en, connected in a single unit by successive partial overlapping, placed in a plastic frame with two holes, one of which is for applying the sample, and the other is for observing the results, and made with the possibility of detecting impurities that impede immunochromatographic analysis, contains bags with a disposal function waste analysis, their isolation from the external environment and sterilization, with each set contains all the necessary functional components

Description

Application area

The utility model relates to stackings for a specific indication of microorganisms and toxins. The laying of immunochromatographic indicator elements is intended for sampling from the external environment and identifying causative agents of especially dangerous infectious diseases (OOIs): anthrax, plague, tularemia, glanders, as well as type A botulinum toxin by immunochromatography.

State of the art

The so-called simple, widely known and very profitable “Reagentless methods” of analysis, working with visual or simple instrumental registration, covering a wide range of microorganisms and toxins. One of these methods is immunochromatography. The specificity of the method is due to the specific immunochemical interaction between antibodies and antigens of microorganisms. Structurally, a typical test system (indicator element) is made in the form of a plastic mandrel 2 × 5 cm in size with windows, inside of which there are polymer membranes with immunoreagents applied to them. For safety, the plastic frame with a membrane and a disposable pipette are sealed in a multilayer plastic bag with a silica gel desiccant. During the analysis, the package is opened. A liquid test sample in an amount of 100-200 μl is applied using a disposable pipette. In foreign scientific and technical literature, the name is sometimes used - "manual immunochromatographic analysis" (HHAs - Hand-Held Immunochromatographic Assay).

The most advanced in the field of creating immunochromatographic methods and systems for the specific indication of biological agents (BA) are US firms. So, for example, Osborne Scientific created the Biowarfare Agent Detection Devices (BADD) laying, which allows detecting anthrax pathogens, botulinum toxin, and ricin in environmental objects. Stacking is a suitcase equipped with

hermetically sealed immunochromatographic tests for each type of BA and simulation tests designed for staff training. Analysis time is 3-30 minutes, zero probability of false positive results when conducting 900 tests, shelf life is 2 years at storage temperature + 20 ° + 50 ° C. The operating temperature range is positive.

Tetracore LLC together with Alextex Technology Inc. offers laying the Guardian Bio Threat Alert System (biological threat alert kit “Sentinel”), consisting of immunochromatographic tests to detect spores of the causative agent of anthrax, ricin, botulinum toxin, plague, tularemia, brucellosis, staphylococcal enterotoxin type B, smallpox virus. The styling also includes simulation training tests for personnel, a device for collecting samples, a reader of results that allows you to raise the level of sensitivity and objective evaluation of the results of the analysis, a network voltage adapter for it, a printer. At the same time, service programs are offered that provide for the replacement of expired test systems, staff training, and a 3-year warranty on the results reader.

New Horizons Diagnostics Corp. (USA) offers, based on the principle of immunofocusing of colloidal gold-labeled antibodies on membranes, the SMART test (Sensitive Membrane Antigen Rapid Test - sensitive membrane test for antigen detection). This type of immunochromatographic analysis involves the stage of interaction of gold-labeled antibodies with the desired antigen in the liquid phase at the sample preparation stage, the resulting antibody-gold-antigen conjugate complex interacts with the membrane with immobilized secondary antibodies. As a result of an immunochemical reaction on the membrane, gold-labeled antibodies that react with the antigens of biological agents (BA) are focused as a bright red dot on the membrane. To identify spore forms, fiberglass membranes without secondary antibodies are used. To evaluate positive results, a reflected light photometry device may be used. It is noted that the method is easily automated, and membranes containing reagents can be made to indicate a wide range of BA.

SMART tests for anthrax spores were used by the US military during Operation Desert Storm in 1991. Later, for the same

of the pathogen, a classical immunochromatographic test system, SMART-2, was developed. Advantages of the immunochromatographic method for BA indication are: speed of analysis; the possibility of creating immunochromatographic tests for any type of human-damaging agents of biological origin - bacteria, rickettsia, viruses, toxins; the relative cheapness and the ability to supplement styling with newly created test systems for the detection of other microorganisms or toxins; lack of additional stages of analysis, liquid reagents; the ability to conduct analysis with minimally trained personnel; the possibility of both visual and instrumental recording of the result; good safety and "built-in" quality control of immunochromatographic test systems; the detection threshold for model mixtures approaching traditional solid-phase immunochromatographic analysis.

All existing known styling are functionally identical. Those. they analyze a sample taken from the external environment by taking smears, collecting unknown suspicious powders and drops of liquid, and then dissolving them in the so-called "Analysis buffer" (a certain composition of an aqueous solution of salts, detergents and protein) and transferring part of the resulting solution (or suspension of particles) to the indicator element (sometimes called a test strip). Then, after passing the reaction, colored bands appear on the surface of the indicator element, which indicate the presence of the desired microorganism, toxin, etc. substances in the sample. The absence of bands indicates the absence of the desired analyte.

Indicator elements (test strips) must be strictly specific, i.e. the indicator element for detecting anthrax should identify only the causative agent of anthrax, and no other microorganisms or toxins.

However, the sample from the external environment may contain: natural acids, alkalis, very fine dust particles, detergents and reducing agents and oxidizing agents, which can interfere with the course of immunochemical reactions and cause the appearance of colored bands on the surface of the indicator element (test strip) even in the absence of the desired biological agent. This phenomenon is called the “false positive result” of the analysis.

The phenomenon of a “false positive result” is inherent in all known styling.

Another disadvantage of the known solutions is that they contain microtampon from

non-woven polymer material. When processing hard surfaces to collect microorganisms and toxins, these materials are ineffective. Another disadvantage of the known solutions is that each immunochromatographic element has its own microtampon for collecting the sample and its own analysis buffer with a volume of about 0.4 ml intended for collecting the sample. Sampling and analysis on each immunochromatographic indicator element occurs sequentially, which lengthens the analysis procedure.

The technical result of the claimed utility model is to shorten the analysis procedure, incl. in the process of studying washouts from hard surfaces, and the exclusion of "false positive results" of analyzes.

Brief Description of the Drawings

Figure 1 shows a constructive stacking device, where 1 - kits for sampling, 2 - a kit for developing operator skills, 3 - kits for analyzing a sample, 4 - pencils, scissors, tweezers, a glass recorder, 5 - a package for sterilization of waste, latex gloves, 6 - operating instructions, 7 - stacking case, 8 - nameplate, 9 - transport packaging (cardboard box), 10 - seal. Figure 2 shows the appearance of the styling.

Utility Model Implementation

The claimed technical result is achieved due to the fact that the laying of immunochromatographic indicator elements, containing inside the hermetically sealed enclosure: kits for sampling, kits for analysis of the sample, pencils, scissors, tweezers, a glass recorder, a bag for sterilization of waste, latex gloves, nameplate, shipping container , characterized in that it contains a hair brush in the sampling kit, contains at least one indicator immunochromatographic element, which is a set of membranes an, integrally connected in the overlap by sequentially placed into a plastic frame with two openings, one of which for application of the sample, and another - for the observation results, and adapted to identify the impurities that hinder the conduct immunochromatographic assay,

contains bags with the function of utilizing analysis wastes, isolating them from the external environment and sterilizing, with each kit containing all the necessary functional components for analyzing a single sample. In addition to indicator immunochromatographic elements used to identify causative agents of plague, anthrax, tularemia, glanders and botulinum toxin type A, each stacking kit analysis kit contains one indicator immunochromatographic element (IIHE) to identify impurities that interfere with immunochromatographic analysis. The IEEE for identifying false positive results of analysis is a set of membranes joined together by successive partial application (multi-membrane composite), placed in a plastic frame with two holes: a hole for applying a sample and a hole for observing the results. A multi-membrane composite consists of (in the sequence of attachment of the membranes to each other): an absorbing membrane of porous cellulose for applying the test sample (A), a fiberglass membrane (B), impregnated with a solution of colloidal gold conjugate with monoclonal antibodies (AT1) against certain microorganisms, nitrocellulose the membrane (B), on which the antibodies are applied, in the form of two narrow bands located at a certain distance from each other. The first lane (analytical zone) contains monoclonal antibodies (AT2), the second lane (control zone) rabbit anti-mouse antibodies. At the same time, AT2 monoclonal antibodies do not react with microorganisms or toxins to which AT1 monoclonal antibodies are specific. A porous membrane is attached to the end of the nitrocellulose membrane (B) to absorb liquid (D). The principle of the IEEE to identify impurities that interfere with the analysis is that: a liquid sample is applied to the membrane to absorb the sample (A), under the action of capillary forces, a liquid with impurities dissolved in it penetrates the membrane (B), on which the colloidal conjugate is located gold with AT1 and dissolves it, the resulting solution by capillary forces moves along the nitrocellulose membrane (B) to the applied antibody bands, and is then absorbed by the membrane to absorb liquid (D).

In the event that the sample contains aggressive chemical impurities of an acid, alkaline, redox nature, or mechanical impurities in

In the form of nanoscale dust particles in high concentration, the conjugate of colloidal gold with antibodies is partially destroyed, while colloidal gold is deposited in the analytical and control zones of the nitrocellulose membrane (B) as colored bands. The appearance of these bands indicates the presence in the test sample of aggressive chemical or nanoscale mechanical impurities that can affect the formation of immunochemical complexes [antibodies-microorganism or toxin], thereby causing a false positive result. In the absence of these impurities in the sample, the process of moving the liquid sample described above occurs without destroying the conjugate of colloidal gold with antibodies and, as a result of the immunochemical reaction of the conjugate of colloidal gold with mouse antibodies and rabbit anti-mouse antibodies, a colored complex forms in the control zone of the membrane [B] [ antibody-colloidal gold-anti-species antibody] observed by the operator as a colored strip in the IIHE control zone.

The use of IIHE increases the reliability of the analysis, by eliminating false positive results. Increasing the reliability of the analysis entails economic consequences, as excludes costly measures to disinfect the area, transport or isolate people who come into contact with objects infected with microorganisms and toxins.

Stacking can be additionally completed with newly developed immunochromatographic indicator elements.

The “Kit for sampling” styling includes a hair brush, which effectively collects microorganisms and toxins on microscales of the hair when processing hard surfaces.

In stacking, a universal analysis buffer is used, i.e. it is suitable for all types of indicator elements included in it (in well-known analogues - each test strip has its own analysis buffer, which differs in chemical composition).

The analysis procedure in the installation includes sampling the external environment and adding it to a significant volume of the analysis buffer (1.0 ml) and then adding aliquots of 120-150 μl to 6 different indicator immunochromatographic elements in parallel. Thus, after 20-30 minutes of the immunochemical reaction, the operator receives the result immediately on 5 types of microorganisms and toxins and is convinced

the absence of impurities that can cause false positive results of the analysis. In laying applied the modular construction principle. The stack contains:

- a separate set for sampling from the external environment,

- a separate set of indicator immunochromatographic elements,

- a separate set for elements of developing operator skills,

- There are separately bags for the disposal of analysis wastes (their isolation from the external environment and sterilization.

In addition, each kit contains all the necessary functional components. This increases the degree of ease of work with laying, since for the analysis of a single sample, you can not use the entire laying, but only use the set for sampling and the set for analysis of the sample.

Stacking allows you to increase the range of identified pathogens of especially dangerous infections and toxins up to 5 items in a single analysis cycle.

The stacking is operated in stationary conditions or as part of mobile laboratories and is designed to carry out the selection and analysis of 10 samples. Work with styling while driving is not provided. Each installation is operated by one operator.

Scope: identification of pathogens of OOI and botulinum toxin type A in samples taken from environmental objects (washings from the surfaces of various objects), or liquid samples obtained during the cultivation of microorganisms on nutrient media.

The styling case can be made in the form of a plastic case and is intended for storage and transportation of styling components. The kit for developing operator skills is intended for pre-training personnel with basic skills in working with styling and interpreting the results of analysis.

The kit for sampling is designed to take one sample and deliver it to the place of analysis, as well as to take one control sample for subsequent analysis by reference methods.

The kit for analysis of the sample is intended for analysis and contains an indicator immunochromatographic element (IEEE) to identify pathogens of OOI, as well as IEEE to identify impurities that cause

false positive results of immunochromatographic analysis. During operation, auxiliary styling components are used: gloves, scissors, a pencil, a glass recorder, bags for the disposal of biomaterials after analysis, a direction form.

The nameplate is marked on the plastic case with the indication:

- logo of the manufacturer;

- full and abbreviated name of laying;

- numbers of these specifications;

- serial number of laying.

Laying principle

The operator opens the installation, takes out a package with a kit for developing operator skills and opens it with scissors. The kit for developing operator skills with styling may contain IEEE positive control - 3 pieces, IIHE negative control - 3 pieces, a plastic Pasteur pipette, test tube with 2 ml of assay buffer.

The operator takes out one IHE from the package for developing positive operator control skills and one IEE for negative operator control skills, a test tube with an assay buffer, and a plastic Pasteur pipette. Then he opens the packages with IIHE and puts the plastic frames on a flat horizontal surface. Opens a test tube with assay buffer and draws a buffer into the pipette. Checks the time on the stopwatch. It drips into both IIHE one drop into the holes for applying the sample (the hole is marked with the Latin letter “s” on the surface of the plastic frame). After 30-45 seconds, the operator drips another drop in the same holes. In total, 5-6 drops are required in each IIHE at intervals of 30-45 seconds. Analysis results are recorded after 25 minutes.

In the IIHE positive control, two colored lines should form, one in the analytical zone (marked with the Latin letter “t” located on the surface of the plastic frame) and the second in the control zone (marked with the Latin letter “c” located on the surface of the plastic frame).

In IIHE, a negative control should form only one colored line in the control zone.

Before starting work with laying, check the completeness according to the table of the instruction manual. When laying with frost in a heated room, its components must be brought into compliance with the room temperature, in order to avoid condensation of moisture on the external surfaces of the IHE. Immediately before going to the object for sampling, the operator takes out and opens with scissors the required number of bags with the kit for sampling, pencils in the blanks and, if necessary, marks the tubes with the analysis buffer using a marker, as well as takes out the bag for sterilization and, if necessary, tweezers .

Samples of suspected OOI pathogens or type A botulinum toxin are sampled using personal protective equipment (type 1 anti-chemical or anti-plague suits, or rubber gloves, a cotton-gauze bandage that protects the respiratory system, glasses, a dressing gown, shoe covers).

Sampling from environmental objects is carried out from solid surfaces: the surface of tables, floors, walls, screens of monitors and TVs, mirrors, glasses, leaves and tree trunks, surfaces of fruits and vegetables. Sampling is as follows. The operator, arriving at the site, takes out a pre-opened bag with a kit for sampling, takes out a labeled tube with analysis buffer and a brush from it. Periodically, wetting the brush in the analysis buffer and squeezing the excess liquid on the edge of the tube, the operator passes the brush along the surface to be examined. In this case, the cells of the pathogens of OOI or botulinum toxin are transferred to the tube. Liquid samples are taken with a dry brush, squeezing the brush against the inner edges of the tube. The area from which the sample is taken should be approximately 100 cm ² (10 × 10 cm). After completion of sampling, at least 1.2 ml of liquid should remain in the test tube. After selection, the tube is sealed with a cap. The used brush is placed in the bag for the kit for sampling. A sample tube and a sample bag are placed in a bag for sterilizing biological waste and a knot is tied on the bag. In this form, the sample (s) are transported to the place of analysis of the sample. The sampling kit contains a sterile tube and a dry swab for taking a smear and subsequent microbiological analysis. Smears for microbiological analysis must be taken before sampling for immunochromatographic analysis, because liquid analysis buffer for

immunochromatography contains a biocidal substance - sodium azide.

The operator takes out from the stack and opens with a pair of scissors a bag with a plastic Pasteur pipette, a bag for analysis of the sample. Then he takes out packets from IIHE from the bag, opens them and puts them on a flat horizontal surface. Each IIHE is put on the package in which it was packed. Then the operator takes out the previously selected sample and draws it into the pipette.

Sample analysis is carried out as follows. The sample is dripped into a hole in a plastic frame marked with the Latin letter “s”. One drop of the sample is dripped into all IIHE and wait 30-45 seconds. The procedure is repeated. In total, 5-6 drops of the sample are added to each IIHE at intervals of 30-45 seconds. During the analysis, colored lines are formed on the surface of the IIHE membrane in the analytical zone (marked with the Latin letter “t” located on the plastic frame) and the control zone (marked with the Latin letter “c” located on the plastic frame). The result is recorded visually after 25 minutes.

After the analysis, all samples, dishes, pipettes should be placed in a plastic bag for the disposal of biological waste and sterilized at a temperature of + 160 ° C for 60 minutes.

Analysis results are carried out on the following basis. A positive result of the analysis is that two colored lines formed on the membrane surface.

Negative result - one colored line was formed on the membrane surface in the control zone. IIHE unsuitable for analysis - not a single colored line was formed on the membrane surface.

If an OOI pathogen or a toxin is present in the test sample at concentrations beyond the norm, then a positive analysis result will be observed in the corresponding IIHE.

If in IEEE for the detection of impurities that cause false-positive results of immunochromatographic analysis, a positive result of the analysis is observed, then the indications of the remaining IEEE are not considered.

In case of emergency operating conditions, the operator must place the selected samples in a stack, close the plastic case and leave the scene of the accident.

Therefore, the styling body is sealed.

Laying in a shipping container can be transported by any means of transport in

covered wagons or containers at temperatures from plus 5 to plus 35 ° C. In open vehicles, a transport box with styling is fixed with improvised means from movements and impacts, and covered with a tarpaulin from precipitation.

For transportation of the packing and replenishment kit without a transport box (for short distances), additional protection is not required.

The components are made of chemically and biologically inert materials (nitrocellulose, high-impact plastics, steel, polypropylene, inorganic salts) and do not pose a direct health hazard. At the end of the operation of the kit, they are disposed of using generally accepted methods based on environmental requirements.

Claims (1)

A package containing inside the hermetically sealed enclosure sample collection kits, sample analysis kits, pencils, scissors, tweezers, a glass scanner, a waste sterilization bag, latex gloves, a nameplate, transport packaging, characterized in that it contains a hair brush in the sampling kit , contains at least one indicator immunochromatographic element, which is a set of membranes connected into a single unit by successive partial application, placed in a plastic frame with two knowing holes, one of which is for applying the sample, and the other is for observing the results, and made with the possibility of identifying impurities that impede the immunochromatographic analysis, it contains bags with the function of disposing analysis wastes, isolating them from the external environment and sterilizing, each kit contains all necessary functional components for the analysis of a single sample.