RU2441666C1 - Complex antigen of measles virus used as a component of immunoenzymometric test system for diagnostics of antibodies to measles virus - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: complex antigen of measles virus used as a component of immunoenzymometric test system for diagnostics of antibodies to measles virus was produced from culture liquid containing measles virus stain Leningrad 16 with titer no less than 105.0 50% tissue cytopathic dose/ml, Edmonston - no less than 106.0 50% tissue cytopathic dose/ml, NovO/96 no less than 108.0 50% tissue cytopathic dose/ml by inactivation of its infectious activity by detergent and separation of protein from cellular lysate by chromatographic purification in amount no less than 2 mcg/ml with the purity no less than 70%. Culture virus-containing liquid was processed using separate cultivation of measles virus stains Leningrad 16, Edmonston and NovO/96 on monolayer of Vero cells culture with subsequent mixing of culture virus containing liquid in proportion 1:1:1 v/v. ^ EFFECT: Usage of invention provides for increased sensitivity and specificity of a complex antigen possessing the property to detect antibodies in blood serum. ^ 2 cl, 4 tbl, 6 ex

Description

The invention relates to a new complex measles virus antigen for use as a component of an enzyme-linked immunosorbent diagnostic test system and can be used in medical virology and microbiology.

Known measles virus antigen obtained on the basis of a strain of measles virus Leningrad 16 (L-16), used as a component of a diagnostic test system (Manual for vaccine and serum case under the editorship of the Academician of the Academy of Medical Sciences P.N. Burgasov. M .: "Medicine ", 1978. - p. 220-223). Development of the technology for producing measles virus antigen strain L 16 on tissue culture from Japanese quail embryos (Vasilyeva G.A., Boychuk L.M. - “Specific measles prevention.” - Materials of the Pasteur LNIIEM Scientific and Practical Conference. - L., 1970. - p.206-210).

However, this measles virus antigen provides insufficient sensitivity and specificity for detecting antibodies using test systems based on it.

Known measles virus antigen obtained on the basis of a measles virus strain Edmonston (US Patent No. 4211843, IPC A01N 1/02; A61K 39/12, publ. 08.07.1980). Strain Edmonston is deposited in the American Cell Culture Collection (ATCC - VK-24, 4/92). Four passages on the Vero cell culture took place at the SSC VB Vector. The authenticity of the strain was established by the method of positive polymerase chain reaction with specific primers. The strain belongs to genotype A. The maximum titers of the virus reach 5-6 days after infection with a monolayer of Vero cells and are 5 × 10 ⁶ TCPD ₅₀ / ml [Agafonov A., et al. Vopr.virusol., 1997, N.1 p.102-205]. Antigenic properties of the strain: detects antibodies to measles virus in the sera of patients and vaccinated people.

However, this measles virus antigen provides insufficient sensitivity and specificity for detecting antibodies using test systems based on it.

The closest analogue (prototype) is the measles virus antigen obtained on the basis of the measles virus strain NovO / 96 and used as a component of the enzyme-linked immunosorbent assay for the diagnosis of measles virus antibodies (RF patent No. 2230785, IPC C12N 7/00, publ. 20.06 .2004). The virus is produced on a monolayer of Vero cells, the cells are destroyed by freezing-thawing, the cell lysate is obtained by centrifugation and ultrafiltration. The antigen for ELISA is purified from foreign proteins by ultracentrifugation in a sucrose density gradient, which is inactivated by heating at 56 ° C for 30 minutes.

However, this measles virus antigen also provides insufficient sensitivity and specificity for detecting antibodies using test systems based on it.

The technical result of the claimed invention is to increase the sensitivity and specificity of the complex antigen of measles virus, which ensures the detection of antibodies in blood serum.

The specified technical result is achieved by obtaining a complex measles virus antigen used as a component of an enzyme immunoassay for the diagnosis of measles virus antibodies obtained from a culture fluid containing measles virus strains Leningrad 16 with a titer of at least 10 ^5.0 TCD ₅₀ / ml, Edmonston - not at least 10 ^6.0 TCD ₅₀ / ml, NovO / 96, not less than 10 ^8.0 TCD ₅₀ / ml by inactivation of infectious activity of its detergent and protein isolation from cell lysate by chromatographic purification of at least 2 mg / ml with purity not m her 70%.

Virus-containing culture fluid was obtained by separately cultivating measles virus strains Leningrad 16, Edmonston and NovO / 96 on a monolayer of Vero cell culture, followed by mixing of the virus-containing culture fluids in a ratio of 1: 1: 1 by volume.

Antigenic properties of a complex antigen. Detects all specific antibodies to measles virus in the sera of patients and vaccinated people in higher titers than when using each antigen individually (table 1).

Based on a complex antigen, an immunosorbent was created - the main component of a set of single-stage ELISA for the detection of antibodies to measles virus. The set of reagents "Anti-Measles Ig Trisorbent" consists of:

- immunosorbent - a complex antigen of measles virus sorbed in the wells of polystyrene collapsible tablets;

- K1 + - positive control sample No. 1 - human serum containing antibodies to measles virus and not containing antibodies to HIV-1, HIV-2, HCV,

- Treponema pallidum and HBs antigen inactivated by heating at a temperature of 56 ° C for 3 h - 1 vial, 2 ml;

- K2 + - weakly positive control sample No. 2 - human blood serum containing antibodies to measles virus in the minimum reliably detectable amount, not containing antibodies to HIV-1, HIV-2, HCV, Treponema pallidum and HBs antigen, inactivated by heating for 3 h at a temperature of 56 ° C - 1 vial, 2 ml;

- K- - negative control sample - human blood serum that does not contain antibodies to measles virus, HIV-1, HIV-2, HCV, Treponema pallidum and HBs antigen, inactivated by heating at 56 ° C for 3 h - 1 fl ., 3 ml;

- RK - conjugate solution - a solution of mouse monoclonal antibodies to human Ig conjugated with horseradish peroxidase - 1 vial, 11 ml;

- PX - chromogen solution containing TMB - 1 vial, 13 ml;

- FSB-T (× 25) - 25-fold concentrate of phosphate-saline buffer solution with tween - 1 vial, 26 ml;

- stop reagent - 1 vial, 6 ml.

Analytical sensitivity and analytical specificity of the kit were determined in ELISA using a comparative setting of the serum panel, characterized in ELISA using kits for the quantitative determination of IgG for measles virus produced by Vector-Best CJSC and Medical and Biological Union CJSC - VectoKor-IgG and Measles-IgG-DS, respectively. Comparative characteristics of reagent kits for a one-stage ELISA, including the “Anti-Measles Ig Trisorbent”, obtained on the basis of the inventive complex antigen, are shown in table 2.

table 2 Comparative characteristics of a set of reagents for a one-stage ELISA “Anti-Measles Ig Trisorbent” Reagent kit The name of the groups of serum donors of the study panel Not vaccinated, not sick N = 89 Once vaccinated N = 89 Double vaccinated N = 89 Having had measles N = 89 "VektoKory-IgG" 0 / - 63 / 0.47 87 / 1.05 96 / 3.3 "Measles-IgG-DS" 0 / - 89 / 0.59 94 / 3.2 99 / 6.8: “Anti-Measles Ig Trisorbent” 0 / - 90 / 0.67 98 / 5.5 100 / 17.4 Note: Table 2 shows the number of positive samples / average specific activity of positive samples in IU / ml.

From table 2 it is seen that the analytical sensitivity and specificity of the test system "Anti-Measles Ig Trisorbent", obtained on the basis of the inventive complex antigen, is significantly higher than the known analogues.

Example 1. A method of obtaining a complex antigen of measles virus

Obtaining a virus-containing fluid

The original Vero cells are stored in liquid nitrogen and grown by serial passages. As the growth medium using a solution of Needle MEM with 8-10% fetal bovine serum. The cell culture is grown for 3-4 days at an inoculum cell concentration of 5-10 × 10 ⁵ cells / ml of medium and reseeded in a conventional manner.

The corresponding measles virus (strains L-16, Edmonston, NovO / 96) separately infects 1-liter culture vessels with a monolayer of Vero cell culture (multiplicity of infection 1:10). After incubation at 20-25 ° C for 40 minutes, 200 ml of MEM needle medium containing 4.0 ± 0.1 ml of cattle serum, 0.001% trypsin, 200 units / ml of benzylpenicillin and 200 ng are added to the culture vessels / ml streptomycin. After incubation for 5-7 days at (36 ± 1) ° C, the nutrient medium is drained. The measles virus titer, strain L-16, in the fluorescence index is not less than 10 ^5.0 TCD ₅₀ / ml, strain Edmonston - not less than 10 ^6.0 TCD ₅₀ / ml, strain NovO / 96 - not less than 10 ^8.0 TCD ₅₀ / ml.

Obtaining Vero Cell Lysate

During cultivation, the transition of the virus from cell to cell is carried out due to the formation of symplasts without the release of the main amount of the latter in the SCL, therefore, the accumulation of the virus occurs both in the SCL and inside the cells. 5-7 ml of 0.01 M Tris HCl, pH = 7.6 and detergent Triton X-100 are added to the culture vessel to a final concentration of 2-3%. The suspension is incubated for 1 h at (36 ± 1) ° C and stirring, then treated with ultrasound 22 Hz 2 times for 20 seconds. The cell lysate is freed from cell detritus by settling with fluids at 4-8 ° C for 8-12 hours or by low-speed centrifugation (JA-14 rotor J2-21 centrifuge (Beckman, USA)) at 10,000 rpm for 20 min at temperature 4 ° С.

Example 2. Selection of conditions for chromatographic purification of complex antigen

DEAE cellulose (Whatman, Sweden) is prepared according to the recommendations of the manufacturer. The DEAE cellulose column was equilibrated with 20 mM Tris-HCl buffer, pH 7.6. The combined suspension of antigenic material of the three measles virus strains is applied to the column at the rate of 1 ml of suspension per 1 ml of sorbent. After application, the column is washed with the same buffer. The sorbed material is eluted by steps of NaCl concentrations in the same buffer solution until the optical density decreases at a wavelength of 280 nm in the solution resulting from the column to a “zero” value. Each fraction is examined for antigen quality by sensitivity and specificity by ELISA (see table 3).

Example 3. Chromatographic purification of complex antigen

The DEAE cellulose column was equilibrated with 20 mM Tris-HCl buffer, pH 7.6. The combined suspension of antigenic material of the three measles virus strains is applied to the column at the rate of 1 ml of suspension per 1 ml of sorbent. After application, the column is washed with the same buffer containing 0.2 M NaCl. The target complex antigen is eluted with 0.4 M NaCl in the same buffer. The target fraction is monitored for compliance with antigen quality by sensitivity and specificity by ELISA.

Table 3 ELISA results when stating positive and negative samples using immunosorbent made on the basis of different fractions of chromatographic purification of complex antigen NaCl concentration for elution of the antigen fraction from the column O.P. positive sample O.P. negative sample OD (+) / OD (-) Non-absorbable fraction (slip) 0.117 0.256 0.5 0.1 M NaCl 0,075 0.179 0.4 0.2 M NaCl 0.187 0,120 1,6 0.3 M NaCl 1,459 0.121 12.1 0.4 M NaCl 2,453 0.163 16.2 0.5 M NaCl 1,469 0.204 7.2 0.7 M NaCl 0.552 0.192 2.9 1 M NaCl 0.340 0.184 1.8

The yield of highly purified complex antigen with 3 L of fluorescence and cell lysate is 15-20 mg.

Example 4. The manufacture of immunosorbent

An immunosorbent is prepared by adding 0.20 ± 0.06 ml of measles complex antigen after purification on a column (see Example 3) to 1.00 ± 0.01 L of sorption solution. The composition of the solution for sorption: 2.00 ± 0.01 g of sodium carbonate, 0.50 ± 0.01 g of sodium azide, 0.400 ± 0.012 ml of 2% phenolphthalein, purified water - up to 1 liter. The immunosorbent is stirred on a magnetic stirrer for 1 h at a temperature of from 18 ° C to 24 ° C.

Application and sorption of immunosorbent on tablets: for sorption use tablets Nunc Medisorp - "Nunc A / S", Denmark, or similar. The filling volume of the wells is 100-105 μl. Sorption is carried out at a temperature of from 17 ° C to 27 ° C, the sorption time is 18-20 hours

Example 5. The methodology of using the reagent kit for a one-stage ELISA "Anti-measles Ig Trisorbent"

For analysis, human serum or plasma is used. For analysis, 10 μl of sample is sufficient. To detect class G antibodies to measles virus, human blood serum (plasma) can be used either freshly prepared or stored for no more than 7 days at a temperature of 2 ° C to 8 ° C, provided that there is no microbial contamination, or no more than 12 months at a temperature of minus 20 ° C. Freezing of serum (plasma) to a temperature of minus 20 ° C is allowed no more than two times.

To carry out the reaction, 90 μl of a solution is added to all wells of the tablet for sample dilution (composition: 2.00 ± 0.01 g sodium carbonate, 0.50 ± 0.01 g sodium azide, 0.400 ± 0.012 ml 2% phenolphthalein, purified water - up to 1 liter). 10 μl of K1 + is added to the well of plate A1; 10 μl of K2 + are added to wells B1 and C1. 10 μl K - are added to well D1. 10 μl of the studied sera are added to the remaining wells of the plate (final dilution of control and test samples - 10 times). The solution was stirred by pipetting and incubated for 40 min at a temperature of 37 ° C. After incubation, the contents of the wells are removed and the plate is washed seven times with a working solution of FSB-T. 100 μl of chromogen solution is added to each well and the plate is placed for 15 minutes in a dark place at a temperature of 37 ° C. The reaction is stopped by adding 50 μl stop reagent to each well of the plate.

The OD value of the solutions in the wells of the immunosorbent is measured on a spectrophotometer in a two-wave mode: the main filter is 450 nm, the reference filter is in the range from 620 to 700 nm. Test results are considered positive if the OD of the test serum is greater than OPK2 + cf. (the lower limit of the positive values corresponds to 0.28 IU / ml). The test results are considered negative if the OD of the test serum is less than OPK2 + cf. - 20% (the upper limit of negative values corresponds to 0.12 IU / ml).

Ps. calculated by the formula:

Psy. = Opt. × 0.25: (OPK2 + cf.)

where opsiv. - the value of the OD of the studied serum.

Asyv. (antibody titer in ME) is calculated by the formula:

Asyv. = 10 ^{(Psi.-a) ÷ b}

where a and b are constants specific for each series (indicated in the Instruction that accompanies each set).

An example of calculating the data obtained is given in table 4.

Table 4 An example of calculating the titer of antibodies to measles virus in the test serum (OPK1 + cf.) - the average value of OD in wells A1 and B1 2,919 - in the application range (OPK2 + cf.) - the average value of OD in wells C1 and D1 (0.273 + 0.275): 2 = 0.274 - in the application range Lower bound of positive values > (OPK2 + cf.) = 0.274 Upper bound of negative values <(OPK2 + cf.) - 20% = 0.220 OPK- - the value of OD in well E1 0.056 <(OPK2 + cf.) - 20% = 0.220 The above values indicate the possibility of accounting for the results of ELISA Opsv - OD value of the studied serum 1,920 Ps. = Poss. × 0.25: (OPK2 + cf.) 1.920 × 0.25: 0.274 = 1.752 a - value of the constant according to the Appendix 1.4 b - constant value according to Appendix 2.1 (Ps. - a): b (1,752-1,4): 2,1 = 0,168 Asyv. 10 ^0.168 = 1.47 Answer: specific serum activity 1.47 IU / ml

Example 6. The use of the reagent kit "Anti-Measles Ig Trisorbent" based on the inventive antigen in clinical practice

1. During the outbreak of an infectious disease in Blagoveshchensk in 2010, the Anti-Measles Ig Trisorbent reagent kit was used to serologically confirm the clinical diagnosis of measles. In 2 patients, 2–3 days after the onset of clinical signs of the disease and 2–4 weeks later, blood sampling and serum were obtained to serologically confirm the diagnosis of measles. The increase in titers of specific antibodies in paired sera was studied in ELISA using commercial Kits “Melisa Meas-IgM” manufactured by ZAO “MBS” and in parallel using the experimental series “Anti-Meas Ig Ig Tricorbent”, the main component of which is an immunosorbent based on a complex antigen measles virus. The diagnosis of measles was confirmed after PCR with specific primers at the C-terminal part of the gene encoding the measles virus NP protein and sequencing of the amplification fragment.

The Melisa Measles-IgM kit revealed specific IgM for measles virus in both patients. When ELISA was performed on the Anti-Measles Ig Trisorbent kit, these patients also noted the presence of specific IgM for measles virus. When determining the increase in titer of specific antibodies in paired sera using the “Anti-Measles Ig Trisorbent” in both patients, at least a 4-fold increase in specific IgG for measles virus was recorded.

In the samples of patients by PCR, the genetic material (RNA) of measles virus was detected. Sequencing showed that the virus causing the outbreak belongs to genotype N.

Thus, the Anti-Meas Ig Ig Trisorbent reagent kit can be used to detect early antibodies to measles virus, as well as to determine specific antibodies in paired sera obtained from patients, and thus to determine, withdraw or confirm the diagnosis during the course of the disease with clinical manifestations characteristic of measles.

2. Between 2005 and 2010 in the Novosibirsk region work was carried out to study the titer of specific antibodies to measles virus in the blood serum of children aged 6 years before the second vaccination against measles. The titers of specific antibodies were studied in the RTGA reaction (according to the standard method using erythrocytes of the monkey Macaca mulatta) and the ELISA method (using the inventive set of reagents "Anti-Measles Ig Trisorbent").

A comparison of ELISA and RTGA methods for the determination of measles antibodies in the blood serum of children showed that using Anti-Meas Ig Ig Torborbent specific antibodies were determined in 235 sera from 237 studied sera. A similar result (235 sera from 237) was obtained using rtga. The arithmetic mean values of the specific activity of measles antibodies determined using the Anti-Meas Ig Ig Triorbent kit were higher than those determined in rtga (6.2 IU / ml and 3.3 IU / ml, respectively).

Thus, the reagent kit for the one-stage ELISA Anti-Measles Ig Trisorbent can be used to determine the intensity of individual immunity to the measles disease.

Claims (2)

1. A complex measles virus antigen used as a component of an enzyme immunoassay for the diagnosis of measles virus antibodies, obtained from a culture fluid containing measles virus strains Leningrad 16 with a titer of at least 10 ^5.0 TCD ₅₀ / ml, Edmonston - at least 10 ^6.0 TCD ₅₀ / ml, NovO / 96 - at least 10 ^8.0 TCD ₅₀ / ml by inactivating its infectious activity with a detergent and isolating protein from the cell lysate by chromatographic purification in an amount of at least 2 μg / ml with a purity of at least 70%. 2. The complex antigen according to claim 1, characterized in that the culture-containing virus-containing liquid was obtained by separately culturing the measles virus strains indicated in claim 1 onto a monolayer of Vero cell culture, followed by mixing of the culture-containing virus-containing fluids in a ratio of 1: 1: 1 by volume.