RU2724897C1 - Test system for simultaneous detection of humoral markers of human herpes virus infection - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, particularly to clinical laboratory diagnostics. Disclosed is a test system for simultaneous detection of humoral markers of human herpes virus infection, comprising an immunosorbent in the form of test sticks (strips), on each of which in form of separate lines the following antigens are deposited: antigen gG-1 HSV-1; ye antigen gG-2 HSV-2; native VZV antigen; gE VZV antigen; EBV antigenes: nuclear - EBNA (Epstein Barr Nuclear Antigen), early - EA (Early Antigen) and capsid - VCA (Viral Capsid Antigen); gess mosaic CMV antigen containing protein sequences pp150, pp52, pp28 and gB; hes large structural protein (H1) virus (HVV-6) UlN virus; glyboprotein B (gB) of HHV-7 virus; gm mosaic antigen HHV-8 containing ORF65 and ORF8 (K8) protein sequences.EFFECT: invention provides the replacement of several laboratory studies in the EIA format by one diagnostic examination, the possibility of simultaneous detection of G class antibodies to all human pathogenic types of herpes viruses, using a minimum amount of biological material compared to several EIA applications and labour saving by laboratory medicine specialists.3 cl, 3 tbl, 3 ex

Description

Technical field

The invention relates to medicine, in particular to clinical laboratory diagnostics, immunochemistry and for the diagnosis of infections caused by human herpes viruses pathogenic for humans, by the method of linear immune blotting (LIB), which is a type of enzyme-linked immunosorbent assay (ELISA), most used in the diagnosis of various viral, bacterial and other infections.

State of the art

According to modern classification, the Herpesviridae family currently includes about 120 types of DNA viruses, subdivided into 3 subfamilies: a-, (3- and y-herpes viruses [1]. Eight representatives of herpes viruses are pathogenic to humans; from the sub-family of a-herpes viruses, their include: herpes simplex viruses type 1 and 2 (HSV-1 and HSV-2, or herpes simplex virus type 1 and 2 - HSV-1 and HSV-2, or human herpes virus type 1 and 2 - HHV-1 and HHV -2) and chickenpox and herpes zoster virus (BBO, or Varicella-Zoster virus - BBZ, or varicella zoster virus - VZV, or HHV-3), from (3 subfamilies - cytomegalovirus (CMV, or cytomegalovirus - CMV, or HHV-5), type 6 herpes virus (HHV-6), which is the causative agent of sudden exanthema and associated with chronic fatigue syndrome and type 7 herpes virus (HHV-7), also detected in patients with chronic fatigue syndrome; from the subfamily - Epstein-Barr virus (EBV, or Epstein Barr virus - EBV, or HHV-4) and herpes simplex virus type 8 (HHV -8), involved in the occurrence of Kaposi's sarcoma. Animal herpes viruses are also known that can infect humans and cause fatal encephalomyelitis [2].

According to the WHO, herpesvirus-related diseases take 2nd place after influenza as the cause of death from viral infections, and this indicator reaches 15.8% [3]. The high prevalence of herpes viruses in the human population is evidenced by the data of individual epidemiological studies, which established that by the age of 18, more than 90% of urban residents are infected with one or more types of herpes viruses, and in 50% of those infected, clinical relapse of the disease is observed annually, due to the absence protective immunity to them [4-5].

Herpes viruses are very active in people with immunodeficiency states (they are referred to as opportunistic infections), leading to clinical lesions of various organ systems, the development of neuroinfection with a high level of disability (50%) and deaths (20%) [6-7]. Many herpes viruses also have high oncogenic activity, this has been proven for HSV, CMV, EBV, HHV-8 and, possibly, HHV-6 [8].

Representatives of herpes viruses (HSV, CMV) are classified as pathogens of the TORCH group, because when pregnant, they can lead to the development of intrauterine infections, damage to the embryos, death of the fetus and newborns [6-7, 9].

Herpes viruses are able to infect almost all tissues and systems of the host organs, where they continuously or cyclically multiply, providing constant readiness for a clinical exacerbation of the infectious process (persistence). As a rule, herpes viruses persist for life in morphologically and immunochemically altered form in the nerve cells of the regional (in relation to the place of primary introduction) ganglia of sensory nerves (latency).

Herpesvirus infection often proceeds in subclinical forms, or clinical manifestations do not have specific signs that make it possible to establish an etiological diagnosis.

The human immune system responds to infection with herpes viruses by the production of specific antibodies of different classes, but this does not ensure the complete elimination of viruses from the macroorganism. The production of virus-neutralizing antibodies is maintained throughout the subsequent life of a person, sometimes in rather high titers, which prevents the manifestation of infection, but does not prevent the occurrence of clinical relapses when the immune defense is weakened [10-12].

The spectrum of clinical manifestations of herpesvirus infections depends on the localization of the pathological process, its prevalence, the state of the patient’s immune system and the antigenic subtype of the virus. Diseases caused by HSV, CMV and EBV are considered as AIDS-indicative due to their frequent detection in this pathology; It has been established that many HHVs can activate HIV, which is at the stage of the provirus, and are cofactors of the progression of HIV infection and AIDS [13].

In recent years, various aspects of the epidemiology, clinic, and prevention of infections caused by relatively little-known human herpes viruses have been actively studied: HHV-6, HHV-7, and HHV-8 [14]. It is assumed that HHV-6 is involved in the etiology of cancer, chronic demyelinating diseases of the central nervous system, multiple sclerosis, chronic fatigue syndrome, and sudden exanthema in children [15].

Modern laboratory diagnosis of herpesvirus infections in humans includes: virological, molecular biological and serological methods, each of which has its own indications and limitations in use. An immunochemical study of antibodies to herpes viruses in ELISA is especially important when examining people with reduced immune defense (during therapy with cytostatics, systemic corticosteroids, with radiation therapy, as well as in people with HIV infection). The most common research method available for most clinical laboratories is an enzyme-linked immunosorbent assay, which allows you to fairly reliably assess the presence of a humoral response of the body to a current or history of herpes virus infection or a combination of them [16]. One of the modern ELISA options is immune blotting - detecting the presence or absence of antibodies to individual pathogen antigens, which allows not only the fact of infection to be detected, but also to evaluate the stage of infection.

The closest analogues to implementation are immunosorbents for linear IB containing specific immunodominant antigens of one or several pathogens adsorbed on the solid phase.

The prototype of the invention is a test system containing a multi-antigenic immunosorbent for linear immunoblotting, which simultaneously includes several specific antigens of different types of pathogens of TORCH infections ("Line-Blot TORCH-Profile"): cell parasite {Toxoplasma gondii) and viruses {Rubella virus, CMV , HSV-1 and HSV-2), deposited in the form of antigenic lines on test strips of nitrocellulose membrane (strips).

The following purified native and recombinant antigens of the main causative agents of TORCH infections were selected for application to the nitrocellulose membrane in order to obtain an immunosorbent:

~ native toxoplasma antigen (Toxoplasma gondii) and recombinant analog of p30 toxoplasma antigen;

~ native rubella virus antigen (Rubella virus);

~ recombinant analogue of the mosaic antigen of cytomegalovirus (CMV), containing immunodominant sequences of proteins pp150, pp52, pp28 and gB;

~ Recombinant analogues of the herpes simplex virus type 1 G1 antigen (HSV-1) and the herpes simplex virus G2 antigen type 2 (HSV-2). In addition, on the immunosorbent are applied:

~ WHO international standard Anti-Rubella Immunoglobulin, Human (NIBSC, code RUBI-1-94), which is a class G human immunoglobulin for rubella virus - at a concentration of 15-20 IU / ml (line "Rub. Cut off);

~ three control lines, two of which ("0.5+" and "2.0+") contain human immunoglobulins of class G or M in different concentrations to calibrate the results of the study, and one line (Quo - control of sample application) contains antibodies to human IgG / IgM to control sample application [17].

The disadvantage of the prototype is the use of antigens of only three types of herpes viruses, which does not cover the entire spectrum of causative agents of human herpesvirus infections. To obtain more complete information about serological reactivity for all 8 types of herpes viruses that are pathogenic for humans in a subject, at least 5 additional studies in ELISA format are required, with each corresponding set of reagents having calibration features for indicators of analytical and diagnostic sensitivity and effectiveness.

Disclosure of the invention

An object of the invention was the development of a test system for the simultaneous detection of humoral markers of human herpesvirus infection and a method for its preparation, the test system containing an immunosorbent in the form of test strips (strips) on which antigens of the main human herpes viruses are applied and immobilized, which allows in the format of linear immunoblotting, conduct a clinical laboratory study of samples of human biomaterial with simultaneous identification of specific humoral markers of infection with each of the 8 types of herpes viruses (class M or G antibodies).

This task is achieved by selecting the most specific human herpesvirus antigens, determining the optimal sorption concentration of each antigen to develop a technology for their application and immobilization on nitrocellulose membrane test strips intended for subsequent use in linear immunoblotting.

The new original composition and production technology involves the phased application of calibrated solutions of recombinant and purified native antigens of 8 types of human herpes viruses to the nitrocellulose membrane and providing conditions for their stable immobilization.

Thus, the claimed test system for the simultaneous detection of class G antibodies to antigens of human pathogenic types of herpes viruses by linear immunoblotting, including the immunosorbent in the form of test strips from the nitrocellulose membrane, on each of which the following antigens are applied in separate lines:

ges antigen gG-1 HSV-1;

ges antigen gG-2 HSV-2;

native antigen VZV;

ges antigen gE VZV;

HES EBV antigens: nuclear - EBNA (Epstein Barr Nuclear Antigen), early - EA (Early Antigen) and capsid - VCA (Viral Capsid Antigen);

hece mosaic CMV antigen containing protein sequences pp150, pp52, pp28 and gB;

hes large structural protein U11 of the HHV-6 virus;

hec glycoprotein B (gB) of the HHV-7 virus;

HES mosaic antigen HHV-8, containing the protein sequences ORF65 and ORF8 (K8).

In addition, in addition, on each test strip there are areas for marking, in particular, for the identification number.

In addition, each test strip also contains a control line - sample introduction control (Quo), designed to control the introduction of a biological sample, represented by anti-species antibodies to human IgG.

The technical results of the claimed invention are:

replacement of several laboratory tests in the ELISA format to determine specific class G antibodies to each type of herpes virus in biological samples (up to 8-9 independent ELISA studies) with one diagnostic study in the format of linear immunoblotting;

unification of laboratory test methods for the intensity of the humoral immune response to each antigen of a certain type of herpes virus included in the immunosorbent;

the possibility of simultaneous detection of class G antibodies to all types of herpes viruses that are pathogenic for humans, which is achieved through the use of antigens of all 8 human herpes virus infections pathogens in the immunosorbent;

the use of a minimum amount of biological material in linear immunoblotting compared to several ELISA assays;

saving labor costs by laboratory medicine specialists when replacing 8-9 ELISA assays with one study in linear immunoblotting format.

In the process of studying scientific literature (both Russian and foreign) on herpes viruses that are pathogenic for humans, an idea was formed which of the antigens are of the greatest diagnostic value.

When studying the literature on herpes simplex viruses of type 1 and 2, it was found that the human humoral response is primarily generated to the type-specific antigen gG (gG-1 HSV-1 and gG-2 HSV-2) [18, 19], with cross-reactivity between gG-1 HSV-1 and gG-2 HSV-2 has not been established [20]. Therefore, these antigens began to be used to create enzyme-linked immunosorbent assay systems designed to diagnose the infection caused by HSV-1 and HSV-2 (herpetic infection), including the differentiation of these two types of HSV [21].

These literature served as the basis for the inclusion of the recombinant gG-1 HSV-1 and gG-2 HSV-2 recombinant antigens in the developed test system.

When studying the literature on the Varicella-Zoster virus, it was found that this virus contains several highly specific antigens (gH, gE, gB, gL), the detection of antibodies to which has equally significant diagnostic value [22, 23]. Based on these data, it was decided to include in the composition of the immunosorbent the developed test system of the antigenic line represented by the native BBZ lysate (antigen) containing all the specific proteins of the virus. However, due to the fact that all herpes viruses are similar in structure, cross-reactions may occur when antibodies to individual species of the family are detected. Thus, it is known that antibodies to certain varicella zoster antigens can cross-react with the herpes simplex virus antigens of the first type [24]. An exception is the BBE gE antigen [25]. In this regard, in order to differentiate the BBZ from HSV-1, it was decided to include in the immunosorbent an additional line represented by the BBZ gE antigen.

When studying the literature on the Epstein-Barr virus, it was found that IgG to the nuclear antigen (EBNA - Epstein Barr Nuclear Antigen) appears in human blood a few weeks after the onset of clinical signs and persist throughout life. Early antigen IgG (EA - Early Antigen) appears from the first week of illness and serves as a marker of acute infection. IgG to the capsid antigen (VCA - Viral Capsid Antigen) usually appears (but not always) a few weeks after infection and lasts for life [26]. These data served as the basis for the inclusion in the composition of the immunosorbent of all three of the above antigens of the Epstein-Barr virus.

When studying the literature on cytomegalovirus, it was found that the most diagnostically significant antigens are the phosphoproteins pp150, pp52, pp28 and the glycoprotein gp55 (gB) [27]. IgG to pp150 is usually produced in every case of CMVI, and as the infection progresses, IgG to protein pp28 appears. The presence of IgG to pp52 and gB antigens is evidence of a long-past (more than 6 months) infection [28]. Based on these data, it was decided to include in the composition of the immunosorbent a line represented by a mixture of all significant recombinant CMV antigens (pp150, pp52, pp28 and gp55 (gB)).

When studying the literature on human herpes simplex virus type 6 (HHV-6), it was found that HHV-6 is of two types: HHV-6A (neurovirulent) and HHV-6B (causing the disease "baby roseola") [29, 30, 31] . The most immunogenic antigen is an antigen with a molecular weight of 101 kilodaltons (kDa), homologous to both types of the virus and lacking cross-reactivity with HHV-7 [32]. This antigen was selected for the application of the HHV-6 line on the immunosorbent.

When studying the literature on human herpes simplex virus type 7 (HHV-7), it was found that glycoprotein B (gB) is highly conserved among individual strains of HHV-7 and induces a specific human immune response. In addition, this antigen does not cross-react with antigens of other herpes viruses [33]. On this basis, glycoprotein B (gB) was selected for inclusion in the immunosorbent.

When studying the literature on human herpes simplex virus type 8 (HHV-8), it was found that the most diagnostically significant antigens are the proteins ORF65 and ORF8 (K8). As HHV-8 infection develops, IgG titers to ORF65 increase. The use of K8 antigen provides high sensitivity and specificity of the study, since K8 does not have a homolog among other herpes viruses, which means there is no potential cross-reaction with antibodies against other herpes viruses [34]. In this regard, it was decided to include in the composition of the immunosorbent a line represented by a mosaic antigen containing the immunodominant sequences of the ORF65 and ORF8 (K8) HHV-8 proteins.

The implementation of the invention

The essence of the invention is to obtain a diagnostic test system by manufacturing an immunosorbent that includes diagnostically significant antigens of 8 types of human herpes viruses (ges antigen gG-1 HSV-1; ges antigen gG-2 HSV-2; native antigen VZV; ges antigen gE VZV; ges EBV antigens: nuclear - EBNA, early - EA and capsid - VCA; hemes mosaic CMV antigen containing sequences of proteins pp150, pp52, pp28 and gB; hes large structural phosphoprotein Ul1 of the HHV-6 virus; heces glycoprotein B (gB) of the HHV- virus 7; hemes mosaic antigen HHV-8, containing the sequences of proteins ORF65 and ORF8 (K8)), preparation of a solution for diluting samples - PPO, 10-fold washing solution concentrate - PRxU, conjugate, as well as a complete test system.

Short description of tables

Tab. 1. The intensity of staining of antigenic lines.

Tab. 2. The sensitivity study of the developed test system “Line-Blot HHF-profile-IgG” in comparison with the test system-prototype “Line-Blot TORCH-Profile-IgG”.

* The test system-prototype “Line-Blot TORCH - Profile-IgG” does not imply the detection of IgG for VVZ, VEB, HHV-6, HHV-7, HHV-8 (highlighted in gray)

Tab. 3. The study of the specificity of the developed test system “Line-Blot HHF-profile-IgG” in comparison with the test system-prototype “Line-Blot TORCH - IgG Profile”.

The invention in particular cases is illustrated by the following examples.

Example 1

1. The manufacture of immunosorbent.

An immunosorbent (strips from a nitrocellulose membrane) is prepared by direct application of the antigens HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, HHV-8 on a nitrocellulose membrane.

1.1. Membrane preparation.

Prepare the required number of nitrocellulose membranes measuring 10x20 cm.

Prepare a 10-fold concentrate of phosphate-saline buffer solution (FSBx10).

Composition per 1 liter of solution:

- sodium phosphate 2-substituted 12-aqueous (Na ₂ HPO ₄ x12H ₂ O) -9.3 g;

- sodium phosphate 1-substituted 2-aqueous (Na ₂ HPO ₄ x2H ₂ O) -0.65 g;

- sodium chloride (NaCl) - 59.5 g;

- sodium thiolate - 0.7 g;

- purified water of the 2nd stage - 1000 ml.

Using a measuring cylinder, add 500 ml of purified water to the flask. Add salt samples to the water tank, mix on a magnetic stirrer until completely dissolved, bring the solution volume to 700 ml with purified water, mix for 10-15 minutes.

Measure the pH of the solution, (the desired pH value is 7.3 + 0.2). If necessary, adjust the pH with a 2 molar solution of sodium hydroxide to the desired value.

Prepare a single dilution of FSBx1 by diluting FSBx10 with purified water, based on the need for 50 ml of FSBx1 per membrane.

Pour the prepared solution of 500 ml each into trays and place 9 nitrocellulose membranes there. in 1 tray. Incubate on a rocking shaker for 15 minutes. Next, put the membranes on filter paper and dry in a laminar cabinet for 55-65 minutes at a temperature of 18-24 ° C or leave on a work table and dry for 80-100 minutes at a temperature of 18-24 ° C.

1.2. Preparation of working dilutions of HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, HHV-8 antigens and anti-IgG antibodies.

Prepare carbonate-bicarbonate buffer (KBB).

Composition (per 1 liter of solution):

- sodium carbonate - 1.6 g;

- sodium carbonic acid - 3.0 g;

- sodium azide - 0.2 g;

- purified water of the 2nd stage - up to 1000 ml.

Using a measuring cylinder, add the calculated volume of purified water to the flask, add salt samples to the container, mix on a magnetic stirrer until the salts are completely dissolved. Check the pH of the solution, the norm is 9.4-9.6. If the pH of the solution does not match this value, prepare the KBB again.

Based on the initial protein concentration of antigens, the working concentration of antigens and the total volume of working dilution of each antigen (depending on the number of membranes), calculate the volume of KBB and the amount of each antigen needed to prepare the given volumes of working dilutions.

Calculation example:

The initial protein concentration of antigen is 3 mg / ml

The volume of working dilution of antigen in the KBB is 2000 μl (for 36 membranes).

The working concentration of antigen is 0.1 mg / ml.

2000 × 0.1 / 3 = 66.7 μl of antigen should be taken.

2000-66.7 = 1933.3 μl KBB should be taken.

In the same way, prepare a working dilution of antibodies against human IgG (control of the introduction of the sample) in a solution of KBB.

Work dilutions prepare immediately before sorption.

1.3. Application of dilutions of antigens on the membrane (sorption)

The application of working dilutions of antigens to the membrane can be done manually using a capillary-piston device or using a machine for capillary lines.

For manual application, fill the capillary-piston device with a working dilution of antigen and, using a ruler, apply a strip of this dilution to the membrane in accordance with the standard scheme for applying antigens HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, HHV-8 and Quo control line (sample entry control).

The standard scheme for applying the antigens HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, HHV-8 and the control line Quo on the strip:

Place membranes with antigens on the shelves of the refrigerator, covered with filter paper and incubated at a temperature of 2-8 ° C for 18-24 hours.

1.4. Membrane blocking Prepare blocking solution (BR). Composition (per 1 liter of solution):

- BSA - 10.0 g;

- FSB × 10 - 100 ml;

- purified water - 900 ml.

Pour BR into 500 ml trays. Gently transfer there with tweezers membranes coated with antigens of 9 pcs.

Place the tray on a shaker-rocker and stand for 55-65 minutes at a pumping intensity of 40 rpm and a temperature of 18-24 ° C, then wash the BR membranes with clean water until the foam disappears on their surfaces.

1.5. Membrane drying and strip cutting

For preliminary moisture removal, gently place the washed membranes for 5 minutes on twisted sheets of filter paper at a temperature of 18-24 ° C with tweezers. Cut each membrane with strips of 3 mm wide using a special cutter, leaving its upper part uncut for marking strips.

The incised membranes should be dried in a laminar cabinet for 3-3.5 hours at 18-24 ° C.

48-52 strips are obtained from each membrane, which represents 2 immunosorbents of 24 strips. Using adhesive tape with serial numbers applied on it (marking), mark the obtained strips, for which the marking is glued to the uncut part of the membrane so that each marking figure falls in the middle of the marked strip, and then cut off the upper part of the membrane above the marking. Cut the marked strips with scissors. Strips of one membrane with tweezers carefully placed in 24 pieces (No. 1-24) in a plastic container test tube.

1.6. Control of immunosorbent in IB

To control each new series of immunosorbent in IB by examining the sera of standard panels containing and not containing antibodies to antigens HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, HHV-8. Consider the new series of immunosorbent suitable for use when the results of the study of the samples correspond to their passport characteristics.

1.7. Labeling and storage of immunosorbent

Stick a label on each test tube with strips indicating the information required by the specifications for the kit. Before completing the kit, store tubes with immunosorbent at a temperature of 2-8 ° C for no more than 12 months.

2. Preparation of a solution for breeding samples (PPO)

2.1. Preparation of a 10-fold concentrate of Tris-saline buffer solution (TSBh10)

Composition (per 1 liter of solution):

- tris- (hydroxymethyl) -aminomethane - 24.20 g;

- sodium chloride - 234.0 g;

- sodium azide - 0.10 g;

- tween-20 - 10.0 ml;

- purified water of the 2nd stage - up to 1000 ml.

Using a measuring cylinder, add purified water in a flask equal to 0.8 of the specified volume of the solution, add weighed portions of tris (oxymethyl) aminomethane and sodium chloride, mix until the reagents are completely dissolved, check the pH. The pH should be 7.4-7.6. If necessary, adjust the pH with concentrated hydrochloric acid (7-20 ml of acid is consumed per 500 ml of solution). Add a portion of sodium azide to the container with the solution, mix until the salt is completely dissolved; using the pipette dispenser, add the calculated volume of tween-20, bring the solution to the specified volume with water, mix on a magnetic stirrer for 15 minutes. The resulting TSBx10 filter through an anesthetized filter or filter from filter paper.

2.2. Cooking PPO

Composition (per 1 liter of solution):

- TSBh10 - 80 ml;

- casein sodium salt - 3.00 g;

- chloroacetamide - 2.00 g;

- concentrate blocking solution (CBD) - 180 ml;

- normal goat serum - 65 ml;

- paranitrophenol - 0.10 g;

- bromphenol blue sodium salt - 0.10 g;

- purified water - 675 ml;

Using the measuring cylinder, add the calculated volume of TSBx10 to the flask, add casein and chloroacetamide to the container, mix for 15 minutes on a magnetic stirrer until completely dissolved. Using a measuring cylinder, add the calculated volumes of purified water, CBD and normal goat serum to the container, mix for 30 minutes, add a portion of paranitrophenol and bromphenol blue sodium salt to the container, mix for 30 minutes until complete dissolution.

2.3. Filling, labeling and storage of PPO

Conduct PPO bottling, capping and labeling of vials in accordance with the requirements of the specifications for the set. Labeled vials should be stored at 2-8 ° C for no more than 6 months before use when picking.

3. Preparation of 10-fold washing solution concentrate (PRx10)

3.1. Preparation of 10-fold washing solution concentrate (PRx10)

Composition (per 1 liter of solution):

- purified water - 1000 ml;

- tris- (hydroxymethyl) -aminomethane - 24.20 g;

- sodium chloride - 234.0 g;

- sodium thiolate - 0.10 g;

- tween-20 - 10.0 ml

- 4-nitrophenol - 0.10 g.

Using a measuring cylinder, add the calculated volume of purified water to the flask, add weighed portions of tris (oxymethyl) aminomethane and sodium chloride, mix on a magnetic stirrer until completely dissolved, check the pH. The pH should be 7.4-7.6. If necessary, adjust the pH with concentrated hydrochloric acid (10.0-12.0 ml of acid are consumed per 500 ml of solution). Pour merthiolate and 4-nitrophenol into a container with a solution, add the calculated amount of tween-20 using a pipette dispenser, mix for 15 minutes. Filter the obtained PRx10 through an anesthetized filter or filter from filter paper.

3.2. Filling, labeling and storage PRx10

Conduct PRx10 filling on bottles, corking and labeling of bottles in accordance with the requirements of the specifications for the set. Labeled vials should be stored at 2-8 ° C for no more than 6 months before use when picking.

4. Preparation of the conjugate

4.1. Conjugate Preparation

Composition (per 1 liter of solution):

- stabilizing solution for antibodies - 1000 ml;

- conjugate of goat antibodies to human IgG labeled with alkaline phosphatase - 0.03 mg;

- bromphenol blue - 0.001 g.

Using a measuring cylinder, add the calculated volume of the stabilizing solution for antibodies to the flask, pour a sample of bromphenol blue, and use a pipette dispenser to add the goat anti-human IgG conjugate labeled with alkaline phosphatase into the container. Stir on a magnetic stirrer for 15 minutes.

4.2. Conjugate bottling, labeling and storage

Conjugate bottling of bottles, corking and labeling of bottles in accordance with the requirements of the specifications for the set. Labeled vials should be stored at 2-8 ° C for no more than 1 month before use when picking.

5. Filling, labeling and storage of substrate solution (CP)

The substrate solution (5-bromo-4-chloro-3-indolylphosphate and blue-blue tetrazolium) is supplied ready-made. Carry out bottling of CP on bottles, capping and labeling of bottles in accordance with the requirements of the specifications for the set. Labeled vials should be stored at 2-8 ° C for no more than 6 months before use when picking.

6. The complete set of the test system

Assemble the boxes from the cut, place the inserts in them and place the marked components of the test system in the boxes in the following quantities:

Immunosorbent - 24 strips in a plastic container;

PPO-1 fl. (30 ml);

PRx10 - 1 fl. (50 ml);

Conjugate - 1 vial. (45 ml);

CP - 1 fl. (45 ml).

In each box, enclose the instructions for use, the protocol form, the consumer form, tweezers and 3 tablet baths for analysis of 8 cells in a plastic bag.

The authors are not aware of technical solutions having a combination of features similar to the claimed invention. Therefore, the present invention meets the criterion of - novelty.

The use of the most diagnostically significant herpesvirus antigens of the 8 types during immunosorbent production and capsidic - VCA; heme mosaic CMV antigen containing protein sequences pp150, pp52, pp28 and gB; hes large structural phosphoprotein U11 of HHV-6 virus; heme glycoprotein B (gB) of HHV-7 virus; heme mosaic antigen HHV-8 the sequence of proteins ORF65 and ORF8 (K8)) allows you to increase the sensitivity of the analysis with respect to the detection of class G antibodies to herpes viruses, allows you to identify antibodies to the full spectrum of human pathogenic herpes viruses, and also to differentiate different types of herpes viruses by selecting antigens with minimal cross-reactivity.

Test system for the simultaneous multiplex detection of class G antibodies to antigens of the causative agents of human herpesvirus infections: herpes simplex virus types 1 and 2, Varicella-Zoster virus, Epstein-Barr virus, cytomegalovirus, human herpes viruses 6th, 7th and 8- types of linear immunoblotting method "Line-Blot HHV profile" obtained by the proposed method will be widely used in healthcare, in particular in clinical laboratories, for immunochemical and immunological analysis. Therefore, the present invention meets the criterion of industrial applicability.

The method of application of the test system is as follows.

Example 2

Analysis using a test system obtained by the proposed method.

Conducting linear immunoblot analysis

1. Carefully remove the required number of strips (one strip for each test sample) one by one from the container with tweezers and place them in the grooves of the bath with the marked side up. Tightly close the container with unused strips and place it for subsequent storage at a temperature of 2 to 8 ° C.

2. In the grooves of the bath with strips, add 1 ml of PPO, stand for 3-5 minutes while rocking on a shaker-rocker with a swing frequency of 40-50 rpm at a temperature of 18 to 25 ° C.

3. Pipette 20 μl of the studied serum or plasma samples with separate tips.

4. Seal the occupied grooves with adhesive tape or close the lid. Place the bath on the shaker. Incubate for 2 hours at a temperature of 18 to 25 ° C.

5. After incubation, remove fluid from the grooves of the bath, using an automatic pipette or vacuum pump, into a container containing a disinfectant solution.

6. Rinse each strip 4 times with a working (single) washing solution, adding 2 ml of solution to each groove. At the first washing, remove the solution from the grooves, as indicated in paragraph 5, immediately after application. In the next three washes after applying the washing solution, stand the bath for 5 minutes on a shaker. Remove the wash solution as described in paragraph 5.

7. Pipette 1.0 ml conjugate into all used grooves in the bath.

8. Incubate for 30 minutes at a temperature of 18 to 25 ° C on a shaker-rocking chair.

9. After incubation, remove the liquid from the grooves of the bath, as indicated in paragraph 5. Rinse the strips, as described in paragraph 6.

10. Pipette 1.0 ml of substrate solution into all grooves used. Incubate for 15 minutes on a shaker-rocking chair in a dark place at a temperature of 18 to 25 ° C.

11. To stop the reaction, remove the substrate solution, as described in paragraph 5. Rinse the strips 4 times, adding 2 ml of purified water to the grooves and keeping the bath for 1 min on a shaker. Remove water as described in paragraph 5.

12. Place the strips between two sheets of filter paper with the marked side up and stand in a dark place until completely dry, and then register the results.

Registration and accounting of results

The obtained results should be taken into account only on condition that the control line for the introduction of the sample (Quo) is colored. Otherwise, the study must be repeated, since the unpainted Quo line means the absence of a sample.

The presence of antibodies to herpes virus antigens in a human blood serum (plasma) sample is detected by the appearance of colored antigenic bands corresponding to individual herpes viruses.

Depending on the presence and intensity of staining of antigenic lines, the results are divided into positive, uncertain and negative (Table 1).

Upon receipt of an uncertain result, it is recommended to re-examine; if an uncertain result is again obtained in it, it is recommended that blood be taken after 3-4 weeks with a new study to identify antibodies to this pathogen.

Staining of the HES EBV EA antigenic line indicates recent infection of the body with the Epstein-Barr virus.

Example 3

In the department of advanced developments of CJSC EKOlab, experimental series of the developed reagent kit Line-Blot HHF-profile-IgG were prepared.

Tests using the test system of the present invention “Line-Blot HHF-profile-IgG” and the test system-prototype “Line-Blot TORCH-Profile-IgG” showed a higher sensitivity of the first, since the test system prototype does not imply detection of antibodies to Varicella-Zoster virus (BBZ), herpes simplex virus type 6 (HHV-6), Epstein-Barr virus (EBV), herpes simplex virus type 7 (HHV-7) and herpes simplex virus type 8 (HHV-8). The results of the study of the sensitivity and specificity of the developed test system in comparison with the prototype test system are shown in table. 2 and 3.

As a reference material for assessing the sensitivity of the developed test system, we studied the sera of standard panels of positive samples of the company EKOlab CJSC, containing IgG for individual proteins HSV-1 (С0П ⁺ -ВПГ-1-G - 12 samples), HSV-2 ( C0n ⁺ -BPG-2-G - 12 samples), VVZ (COP ⁺ -BB3-G - 12 samples), VEB (COP ⁺ -BEB-G - 12 samples), CMV (COP ⁺ -CMB-G - 12 samples ), HHV-6 (SOP ⁺ -HHF-6-G - 12 samples), HHV-7 (SOP ⁺ -HHF-7-G - 12 samples) and HHV-8 (SOP ⁺ -HHV-8-G-12 samples). These standard panels are made from samples of human blood serum obtained in the commercial laboratory "INVITRO", with a positive result for the presence of IgG for HSV-1, HSV-2, BBZ, VEB, CMV, HHV-6, HHV-7 and HHV-8 respectively. Additionally, the obtained samples were verified in enzyme-linked immunosorbent assay systems manufactured by CJSC EKOlab: IFA-HSV-1-IgG, IFA-HSV-2-IgG, IFA-chickenpox-IgG, IFA-Epstein Barr-IgG "," ELISA-CMV-IgG "," ELISA-HHV-6-IgG "," ELISA-HHV-7-IgG "and" ELISA-HHV-8-IgG, respectively.

As a reference material, when assessing the specificity of the developed test system, we studied the sera of standard panels of positive samples from CJSC ECOlab, which do not contain IgG for individual proteins of HSV-1 (SOP ^- VPG-1-G - 12 samples), HSV-2 (SOP ^- -VPG-2-G - 12 samples), VVZ (SOP ^- -BBZ-G - 12 samples), VEB (SOP ^- -BEB-G - 12 samples), CMV (SOP ^- -CMB-G - 12 samples), HHV-6 (SOP ^- -HHF-6-G - 12 samples), HHV-7 (SOP ^- -HHF-7-G - 12 samples) and HHV-8 (SOP ^- -HHF-8-G - 12 samples). These standard panels are made of samples of human blood serum obtained in the commercial laboratory INVITRO, with a negative result for the presence of IgG for HSV-1, HSV-2, BBZ, VEB, CMV, HHV-6, HHV-7 and HHV-8 respectively. Additionally, the obtained samples were verified in enzyme-linked immunosorbent assay systems manufactured by CJSC EKOlab: IFA-HSV-1-IgG, IFA-HSV-2-IgG, IFA-chickenpox-IgG, IFA-Epstein Barr-IgG "," ELISA-CMV-IgG "," ELISA-HHV-6-IgG "," ELISA-HHV-7-IgG "and" ELISA-HHV-8-IgG, respectively.

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As follows from the table. 2 and 3, according to the results of tests in both test systems, a complete agreement was obtained between the final assessments of the studied samples in relation to the detection of IgG for HSV-1, HSV-2 and CMV. We also obtained a complete coincidence of the estimates of the sera of the standard panels obtained in the study of the developed test system “Line-Blot HHF-profile-IgG” with their initial characteristics.

Thus, by expanding the spectrum of antigens (the inclusion of VVZ, VEB, HHV-6, HHV-7, HHV-8 antigens in the composition of the immunosorbent), the developed Line-Blot HHF-IgG profile test system has higher sensitivity test system-prototype “Line-Blot TORCH-Profile-IgG”, which does not involve the detection of IgG for VVZ, VEB, HHV-6, HHV-7, HHV-8.

Claims (12)

1. 1. A test system for the simultaneous detection of humoral markers of infection with human herpes viruses by linear immunoblotting, including the immunosorbent in the form of test strips (strips), each of which has the following antigens as separate lines: - ges antigen gG-1 HSV-1; - ges antigen gG-2 HSV-2; - native antigen VZV; - ges antigen gE VZV; - HES EBV antigens: nuclear - EBNA (Epstein Barr Nuclear Antigen), early - EA (Early Antigen) and capsid - VCA (Viral Capsid Antigen); -HES mosaic CMV antigen containing protein sequences pp150, pp52, pp28 and gB; - hec large structural phosphoprotein (Large structural protein) Ul1 of the HHV-6 virus; - hec glycoprotein B (gB) of the HHV-7 virus; - HES mosaic antigen HHV-8, containing the protein sequences ORF65 and ORF8 (K8). 2. The test system according to claim 1, characterized in that in addition to the test strip there are sections for marking, in particular the application of the identification number. 3. The test system according to claim 1, characterized in that the test strip contains a control line - control of the introduction of the sample.