RU2784089C1 - Test system for the determination of igm and igg antibodies to metapneumovirus in human sera by enzyme immunoassay - Google Patents

Abstract

FIELD: medicine and virology.

SUBSTANCE: invention relates to medicine and virology. The created test system for enzyme immunoassay of human blood serum, based on the use of a strain of metapneumovirus "NM-1", registered in HCV No. 2476 as a specific antigen accumulated in Hela cell culture and concentrated by differential centrifugation, is suitable for detecting IgM and IgG antibodies and determining their titer to metapneumovirus.

EFFECT: present invention enables to detect a significant increase in IgM and IgG titers and the level of non-immune individuals during serodiagnosis of metapneumovirus in patients. The invention can be successfully used by practitioners, laboratory assistants of medical institutions, especially in conditions of impossibility of PCR diagnostics and to confirm the infection with metapneumovirus.

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Description

The invention relates to medicine and virology and concerns the development of a new original ELISA test system for the detection of antibodies to metapneumovirus, which can be used for diagnostic purposes.

Acute respiratory tract infections are the most common illnesses affecting people of all ages around the world, with children being especially vulnerable. The younger population is disproportionately affected by respiratory infections, which are the leading cause of morbidity and mortality in this demographic. Metapneumovirus infection has clinical manifestations that make it difficult to differentiate from infections caused by other respiratory viruses, especially respiratory syncytial virus (hRSV) [1].

Human metapneumovirus was first isolated from respiratory samples from children with acute respiratory infections of unknown etiology in the Netherlands and Sweden in 2001. It has subsequently been found in people of all ages on all continents. This virus has been identified as a new type of metapneumovirus, human metapneumovirus, which currently belongs to the Metapneumovirus genus of the Pneumoviridae family. Human metapneumovirus (hMPV) is an RNA virus. The hMPV virions are pleiomorphic, spherical, and filamentous in shape. The spherical particles vary in size and have an average diameter of 209 nm. Virion RNA encodes 9 structural and non-structural proteins. Currently, two evolutionary lines (genotypes A and B) and two genetic clusters (subtypes) within each lineage have been identified. The similarity of the nucleotide and amino acid sequences of the isolates of the two main types of hMPV is 80 and 90% [2].

It is known that metapneumovirus is able to penetrate into the central nervous system, leading to the development of severe encephalitis [3]. Metapneumovirus infection is rarely asymptomatic in children. This virus is responsible for 5 to 15% of upper and lower respiratory tract infections. The overall incidence of hMPV infection in adults is lower than in children. Asymptomatic SARS infections are also common in adults, accounting for at least 40% of cases [4]. However, the elderly and immunosuppressed patients can develop severe pneumonia, which can be fatal in some cases. In addition, 6 to 12% of exacerbations of chronic obstructive pulmonary disease in hospitalized patients were associated with metapneumovirus infection [5, 6]. They have common symptoms such as fever, cough, hypoxia, upper respiratory tract infections, lower respiratory tract infections, and wheezing. However, the most common causes of hospitalization are bronchiolitis and pneumonia. The average duration of fever in patients with confirmed metapneumovirus infection is about 10 days. Long-term care facilities report a high mortality rate due to metapneumovirus infection [7]. In young people, metapneumovirus reinfection causes mild symptoms of a cold with fever; in older patients, reinfection can lead to severe manifestations of the disease, such as pneumonitis and even death [8]. In one study, 50% of children with metapneumovirus infection were diagnosed with otitis media, and in another study, metapneumovirus infection was found in about 8% of children presenting to hospital with wheezing. Wheezing is a common clinical symptom seen in numerous studies of children with hMPV-associated lower respiratory tract infections. Metapneumovirus infection can lead to exacerbations of asthma in young children and adults [9].

With the development of the reverse transcriptase multiplex polymerase chain reaction (RT-PCR), a sensitive and rapid assay for the detection of hMPV RNA has been developed. The RT-PCR method has a sensitivity and specificity of 100% and 96%, respectively, and is able to detect co-infections even at very low viral loads that cannot be determined using cell culture or immunofluorescence [10].

A combination of immunofluorescent assays followed by RT-PCR is used to diagnose human metapneumovirus infection [11]. RT-PCR assays are more sensitive for the detection of metapneumovirus RNA than cell culture isolation. However, multiplex assays have reduced sensitivity to a single agent with the convenience of simultaneously detecting several different viruses. Another limitation of molecular detection of all viruses is the ability to detect low levels of viral nucleic acid in the absence of infectious virus [12]. Acute respiratory viral infections (ARVIs) often occur in childhood, and the likelihood of detecting the viral genome before the onset of the disease or for extended periods of time after the resolution of the disease complicates the establishment of a causal relationship of one of several co-discovered viruses.

Humoral immunity factors play an important role in the body's response to metapneumovirus infection, so it is very important to improve immunological methods for the detection of specific antibodies, especially in the early stages of the disease.

As you know, enzyme immunoassay (ELISA) is the main test for the detection of viral antibodies in serum and plasma. ELISA is a universal, serological test used to confirm past infection, the method is considered informative in epidemiological evaluation. Currently known test systems for the detection of antibodies to many viruses. For example, to the respiratory syncytial virus, influenza viruses, adenoviruses, herpes viruses, etc. Recently, a test system for the SARS-CoV-2 enzyme immunoassay has been patented [13].

While infection with hMPV is common, there is no ELISA to detect antibodies to human hMPV. Metapneumovirus infection is common in both children and adults [14, 15] during epidemic and inter-epidemic periods [16]. Most people are likely infected during childhood, but metapneumovirus reinfection occurs throughout life.

Numerous studies of metapneumovirus have generated a large amount of data to understand the molecular basis of its replication, assess the clinical significance and pathogenesis of the disease, however, studies are required to understand the mechanisms of adaptive immunity after infection and confirm the existence of real and long-term circulation of various hMPV genotypes in the human population. The detection of antibodies to hMPV will be of great importance for the diagnosis of hMPV. Therefore, there is a need to develop a test system to identify individuals who do not have specific antibodies that are able to support the development of the epidemic process in inter-epidemic periods. It is necessary to develop a method for the determination of specific antibodies to metapneumovirus, due to the fact that metapneumovirus is detected by PCR with a fairly high frequency annually.

Taking into account the arguments listed above, the present development proposes a new method for the detection of antibodies to metapneumovirus based on indirect solid-phase ELISA.

Brief description of drawings:

In FIG. 1 shows the results of the determination of IgG antibodies and their amount to metapneumovirus in the blood sera of adult patients.

In FIG. 2 shows the results of the determination of IgG antibodies and their amount to metapneumovirus in the sera of children with acute respiratory viral infections.

In FIG. Figure 3 shows the results of the determination of IgG antibodies and their amount to metapneumovirus in the sera of adult patients who recovered from COVID-19.

In FIG. 4 shows the results of the determination of IgG antibodies and their amount to metapneumovirus in the sera of pregnant women.

In FIG. 5 shows the results of the determination of IgM antibodies and their amount to metapneumovirus in the sera of hospitalized children with ARVI.

In FIG. 6 shows the results of the determination of IgM antibodies and their amount to metapneumovirus in the sera of pregnant patients with ARVI.

The problem to be solved by the invention is to develop an indirect enzyme immunoassay method for detecting antibodies to metapneumovirus, based on the use of the metapneumovirus strain "NM-1" as a specific antigen, to create (design) an effective test system.

The technical result from the use of the proposed invention is to expand the arsenal of modern methods used to diagnose viral infections, the proposed test system based on ELISA and designed to detect IgM and IgG antibodies to human metapneumovirus, containing a sorbing specific metapneumovirus antigen obtained from the strain "HM -1", deposited in the State Collection of Viruses (GKV) No. 2476, by accumulating the virus in Hela cell culture, concentrating it by differential centrifugation.

The proposed test system based on ELISA includes:

1. Tablet for enzyme immunoassay with antigen adsorbed in the wells, as an antigen, a strain of metapneumovirus GKV No. 2476;

2. Positive control - human blood serum containing antibodies to IgM and IgG to metapneumovirus;

3. Negative control, human serum, not containing antibodies to metapneumovirus;

4. Conjugate containing a 20-fold concentrate of monoclonal antibodies to human IgM or IgG conjugated with horseradish peroxidase;

5. Chromogen solution containing 0.4% tetramethylbenzidine (TMB) solution

6. Substrate solution containing 0.05 M citrate-phosphate buffer (pH 5.0) with hydrogen peroxide solution (0.015%).

7. Stop reagent;

8. Carbonate-bicarbonate buffer 0.05 M solution (pH 9.25);

9. Wash buffer containing 0.02 M phosphate-buffered saline (PBS) with 0.1% tween-20 solution;

10. Blocking buffer containing 2% solution of bovine serum albumin in PBS;

11. Serum dilution buffer containing 1% solution of bovine serum albumin in PBS.

The test system is stored at a temperature of 2°C to 8°C and a relative humidity of not more than 80%. Freezing of kit reagents is not allowed.

Statistical analysis is carried out using Microsoft Excel software using Student's t-test.

The proposed test system is applicable for the analysis of antibody titer in human blood sera.

To more fully illustrate the present invention, examples are given below. However, these examples should not be construed as limiting in any way the scope of the present invention in all respects.

Example 1. Detection of IgM or IgG antibodies by ELISA and determination of their titer to metapneumovirus by the developed test system.

To obtain virus antigens, a strain of metapneumovirus GKV No. 2476 was used. Virulent strain, intended for research and development of diagnostic test systems. The strain is not contaminated with bacterial microflora, foreign viruses. Virus titer 10 ⁵ TCD ₅₀ /ml. The strain has a high biological, antigenic and immunogenic activity, is characterized by high productivity in sensitive biological cultivation systems. The virus was accumulated in Hela cell culture and concentrated by differential centrifugation. Permissive cells for metapneumovirus are also A549, Hep-2 and LLC-MK2.

- to obtain a sorbing specific antigen of metapneumovirus, metapneumovirus is introduced into the wells of the tablet (CORNING9018, USA) at a concentration of 5.0 μg/ml, 100.0 μl in carbonate-bicarbonate buffer;

- incubation is carried out for 16 hours at 4°C;

- according to the standard procedure, the wells of the tablet are washed at least 3 times with wash buffer;

- a blocking buffer is added (PanEco, Australia);

- incubation is carried out at room temperature for 1 hour;

- according to the standard procedure, the wells of the tablet are washed at least 3 times with wash buffer;

- for enzyme-linked immunosorbent assay, a positive control, a negative control, as well as a series of 2-fold dilutions of the tested samples of human blood sera are introduced into the wells of the tablet;

- incubation is carried out for 2 hours at a temperature of 37°C in a thermostatically controlled shaker with a stirring intensity of 700 rpm;

- according to the standard procedure, the wells of the tablet are washed at least 3 times with wash buffer;

- make a conjugate of monoclonal antibodies to human IgM or IgG with horseradish peroxidase at a dilution of 1:2000 ("JSC Vector-Best", Novosibirsk);

- incubation is carried out for 60 minutes at 37°C;

- according to the standard procedure, the wells of the tablet are washed at least 3 times with wash buffer;

- then make a chromogen-substrate solution;

- subsequent incubation is carried out in a place protected from light for 20 minutes at a temperature of 18°C to 25°C;

- a stop reagent is introduced into the wells of the tablet ("JSC Vector-Best", Novosibirsk);

- measurement of optical density (OD) is carried out at a wavelength of 450 nm against 630 nm on the iMark photometer ("BIO-RAD", Japan).

- analyze the results.

OD in the wells with a negative control sample is calculated from the arithmetic mean value (ODav.K ^- ).

The average value of OD in wells with K ^- should not exceed 0.20 optical density units (OD units) when using the two-wave measurement mode and not exceed 0.25 units. OP when measured on one wave of a wave.

The OD value in the positive control well (K ⁺ ) should be no less than 0.80 units. OP.

Based on the data obtained, we calculate the critical value of the optical density (OPcrit.).

OPcrit.=OPav.K ^- +0.3

The result of the analysis is considered positive if OPb.>OPcrit.

The result of the analysis is considered negative if ODV<0.8×ODcrit.,

where OPBR. - arithmetic mean optical density of three wells with the analyzed serum sample.

Example 2. Detection of IgG antibodies and determination of their titer to metapneumovirus in the sera of adult patients with the proposed test system.

Using the test system, 140 sera samples of adult patients obtained before the COVID-19 pandemic were examined by ELISA for IgG antibodies to metapneumovirus. Statistical analysis was performed using Microsoft Excel software using Student's t-test.

When examining the level of IgG antibodies in the group, it was found that in a titer of 1:400 positive samples was 20.7±3.4% (29 samples). High titers of 1:800 and 1:1600 were detected in 28.6±3.8% (40 samples) and 26.4±3.7% (37 samples), respectively. In a titer of 1:200, the percentage was low 6.4±2.1% (9 samples). Negative sera that did not contain antibodies to metapneumovirus were identified in 17.9±3.2% (25 samples). The results are shown in FIG. one.

Example 3. Detection of IgG antibodies and determination of their titer to metapneumovirus in the sera of children by the proposed test system.

The study of IgG antibodies to metapneumovirus in children diagnosed with acute respiratory viral infections was performed by the proposed test system. The level of IgG antibodies in a group of children aged 2 to 13 years in the amount of 134 sera was studied during the COVID-19 pandemic. The sera are negative for antibodies to COVID-19. Statistical analysis was performed using Microsoft Excel software using Student's t-test.

It was found that in a titer of 1:200 and 1:400 positive samples for metapneumovirus were 17.2±4.3% (23 samples) and 26.9±5.0% (36 samples), respectively. In a titer of 1:800, 16.4±4.3% (22 samples) were found. The smallest number of positive sera was identified in a titer of 1:1600, which amounted to 8.9±3.4% (12 samples), while negative samples were 30.6±5.1% (41 samples). The results are shown in FIG. 2.

Example 4. Detection of IgG antibodies and determination of their titer to metapneumovirus in adult patients who recovered from COVID-19 using the proposed test system.

Using the test system, 23 blood sera samples from adult patients were examined for IgG antibodies to metapneumovirus. Statistical analysis was performed using Microsoft Excel software using Student's t-test.

The results of a serological study showed that a sufficiently high percentage of sera with antibodies to metapneumovirus was determined in a titer of 1:100, which amounted to 39.1±10.2% (9 samples), with a titer of 1:200, 26.1±4.4 % (6 samples). Serum negative for antibodies determined 34.8±9.9% (8 samples). The results are shown in FIG. 3.

Example 5. Detection of IgG antibodies and determination of their titer to metapneumovirus in pregnant patients with the proposed test system.

Serum samples from 101 pregnant patients aged 20-25 years were studied using the test system. Statistical analysis was performed using Microsoft Excel software using Student's t-test.

The determination of antibodies to metapneumovirus showed a high level of positive sera 1:400, which amounted to 27.7±4.4% (28 samples), in titers 1:200 and 1:800 the same percentage of 14.9±3.5% 15 samples), the minimum number of samples with a titer of 1:1600 was determined at 3.0±1.6% (3 samples), sera negative for IgG antibodies 39.6±4.9% (40 samples). The results are shown in FIG. four.

Example 6. Detection of IgM antibodies and determination of their titer to metapneumovirus in the sera of children by the proposed test system.

Using the test system, sera samples of hospitalized children with ARVI were examined for IgM antibodies to metapneumovirus. Statistical analysis was performed using Microsoft Excel software using Student's t-test.

When examining the level of IgM antibodies in 112 sera, it was found that in a titer of 1:100 and 1:400, positive samples for metapneumovirus were 8.9 ± 2.7% (10 samples each). In a titer of 1:200, 15.2±3.4% (17 samples) were found. The smallest number of positive sera was identified in a titer of 1:800, which amounted to 7.1±2.4% (8 samples). At the same time, 59.8 ± 4.6% of negative samples (67 samples). The results are shown in FIG. 5.

Example 7. Detection of IgM antibodies and determination of their titer to metapneumovirus in pregnant patients with the proposed test system.

The proposed test system examined samples of 124 blood sera of pregnant patients with signs of acute respiratory viral infections for IgM antibodies to metapneumovirus. Statistical analysis was performed using Microsoft Excel software using Student's t-test.

The results of a serological study showed that a sufficiently high percentage of sera with the content of IgM antibodies to metapneumovirus was determined in a titer of 1:400, which amounted to 21.8±3.7% (27 samples), with a titer of 1:200, 19.4±3 were determined, 6% (24 samples). In a titer of 1:800, 8.9±2.6% (11 samples) were detected, the minimum number of samples with a titer of 1:1600 was determined at 6.5±2.2% (8 samples). Negative sera amounted to 43.5±4.5% (54 samples). Antibodies in a titer of 1:100 were not detected in the samples. The results are shown in FIG. 6.

Thus, a test system for enzyme immunoassay of human blood serum was created, based on the use of the strain of metapneumovirus "NM-1", registered in GKV No. 2476 as a specific antigen accumulated in Hela cell culture and concentrated by differential centrifugation, suitable for detecting IgM antibodies and IgG and determination of their titer to metapneumovirus. Using the designed test system for metapneumovirus serodiagnosis in patients, it is possible to determine a significant increase in IgM and IgG titers and the level of non-immune individuals.

The development can be successfully used by practitioners, laboratory assistants of medical institutions, especially in conditions of impossibility of PCR diagnostics and for confirming a past infection with metapneumovirus.

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Claims (1)

A test system for the determination of specific antibodies to metapneumovirus in the blood sera of children and adults, based on an indirect variant of solid-phase ELISA, including: a plate for enzyme immunoassay containing adsorbed antigen in the wells - a strain of metapneumovirus GKV 2476; positive control; negative control; conjugate of monoclonal antibodies to human IgM or IgG conjugated with horseradish peroxidase; chromogen solution; substrate solution; stop reagent; carbonate-bicarbonate buffer; wash buffer; blocking buffer; serum buffer.

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