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

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

 

The invention relates to a new complex antigen measles virus for use as a component of immunoassay diagnostic test systems and can be used in medical Virology and Microbiology.

Known antigen measles virus, derived from the strain of measles virus Leningrad 16 (L-16)is used as a component of a diagnostic test system (Guide to vaccine and serum case under the editorship of Academician of AMS Pinboliada. M: "Medicine", 1978. - s-223). Development of technology for antigen measles virus strain L 16 on the tissue culture of embryos of the Japanese quail (Vasiliev GA, Boychuk L.M. - "Specific prevention of measles". Materials of scientific-practical conference of VNIIEM them. Pasteur. - L., 1970. - s-210).

However, this antigen measles virus provides insufficient sensitivity and specificity of antibody detection using test systems based on it.

Known antigen measles virus, derived from the strain of measles virus, Edmonston (U.S. Patent No. 4211843, IPC A01N 1/02; AC 39/12, publ. 08.07.1980 year). Strain Edmonston deposited in the American Cell culture Collection (ATSS - VK-24, 4/92). In SRC VB "Vector" has been four passage on the culture of Vero cells. The authenticity of the installed strain by the method of positive polymerase chain reaction with specific pry the apostrophes. The strain belongs to genotype A. the Maximum titres reach 5-6 days after infection of a monolayer of Vero cells constitute 5×106TCPD50/ml [Agafonov A., et al.// Vopros., 1997, N.1, pp. 102-205]. Antigenic properties of strain: detects antibodies to measles virus in the sera of patients and vaccinated people.

However, this antigen measles virus provides insufficient sensitivity and specificity of antibody detection using test systems based on it.

The closest analogue (prototype) is the antigen measles virus, derived from the strain of measles virus NovO/96 and used as a component of an enzyme immunoassay system for the diagnosis of antibodies to measles virus (patent RF №2230785, IPC C12N 7/00, publ. 20.06.2004,). The virus develop on the monolayer of Vero cells, destroy cells by freeze-thawing, the lysate of the cells obtained by centrifugation and ultrafiltration. The antigen for ELISA clear of foreign proteins by ultracentrifugation in a density gradient of sucrose, which inactivate by heating at 56°C for 30 minutes

However, this antigen measles virus also has limited sensitivity and specificity of antibody detection using test systems based on it.

The technical result of the claimed invention is increased. is a major and specificity of complex antigen measles virus, providing detection of antibodies in serum.

This technical result is achieved by obtaining the complex of antigen measles virus used as a component of an enzyme immunoassay system for the diagnosis of antibodies to measles virus obtained from the culture fluid containing strains of measles virus Leningrad 16 with a titer of not less than 105.0TCD50/ml, Edmonston not less than 106.0TCD50/ml, NovO/96-not less than 108.0TCD50/ml by inactivation of its infectious activity of the detergent and the release of protein from the cell lysate by chromatographic purification of at least 2 μg/ml with a purity of not less than 70%.

Cultural vaccinated fluid obtained during separate cultivation of strains of measles virus Leningrad 16, Edmonston and NovO/96 on the monolayer culture of Vero cells with subsequent mixing of cultural vaccinated liquids in the ratio of 1:1:1 by volume).

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

On the basis of a complex antigen created immunosorbent is the main component of a set of reagents one-step ELISA for the detection of the antibodies is to measles virus. Reagents Anti-Measles Ig Resorbent" consists of:

- immunosorbent - complex antigen measles virus, adsorbed in the wells of polystyrene collapsible tablets;

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

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

- K2+ - slabopolozhitelnym control sample No. 2 serum human blood containing antibodies to measles virus in the minimum reliably detectable quantity that does not contain antibodies to HIV-1, HIV-2, HCV, Treponema pallidum and HBs-antigen, inactivated by heating for 3 hours at a temperature of 56°C - 1 FL., 2 ml;

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

- RK - conjugate solution - a solution of monoclonal mouse antibodies to human Ig conjugated to horseradish peroxidase - 1 FL., 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 FL., 26 ml;

stop-reagent 1 vial, 6 ml.

Analytical sensitivity and analiticheskii what I specificity set were determined in ELISA with a comparative statement panel of sera, characterized in ELISA using kits for the quantitative determination of IgG antibodies to measles virus production company "Vector-best" and CJSC "Medical-biological Union" - "Octocore-IgG and Measles-IgG-DS", respectively. Comparative characteristics of the sets of reagents for one-step ELISA, including "Anti-Measles Ig Resorbent", obtained on the basis of the proposed complex antigen listed in table 2.

Table 2
Comparative characteristics of a set of reagents for one-step ELISA Anti-Measles Ig Resorbent"
The reagent kitThe name of the donor group sera analyzed panel
Not vaccinated, not ill N=89Once vaccinated N=89Twice vaccinated N=89Recover from measles N=89
"Octocore-IgG"0/-63/0,4787/1,0596/3,3
"Measles-IgG-DS"0/-89/0,59 94/3,299/6,8:
"Anti-Measles Ig Resorbent"0/-90/0,6798/5,5100/17,4
Note: table 2 shows the number of positive samples/average specific activity of positive specimens in IU/ml

Table 2 shows that the analytical sensitivity and specificity of the test system "Anti-Measles Ig Resorbent", obtained on the basis of the proposed complex antigen, significantly higher than the known analogues.

Example 1. The method of obtaining complex antigen measles virus

Getting vaccinated liquid

The original Vero cells stored in liquid nitrogen and grown by serial subcultures. As the growth environment of the use of the solution Needle MEME with 8-10% of fetal serum of cattle. The cell culture is grown for 3-4 days at sowing cell concentration of 5-10×105cells/ml of medium and subcultured accepted way.

Accordingly measles virus (strains L-16, Edmonston, NovO/96) separately infect 1 litre culture vessels with monolayer culture of Vero cells (multiplicity of infection of 1:10). After incubation at 20-25°C for 40 minutes in the culture vessels add 200 ml of culture among the s Needle MEM, containing 4,0±0,1 ml bovine serum, 0.001% trypsin, 200 u/ml benzylpenicillin and 200 ng/ml of streptomycin. After incubation for 5-7 days at (36±1)°C pour nutrient medium. The titer of measles virus, strain L-16, CVG is not less than 105.0TCD50/ml, strain Edmonston not less than 106.0TCD50/ml, strain NovO/96 - not less than 108.0TCD50/ml.

Obtaining a lysate of Vero cells

Under cultivation the transfer of the virus from cell to cell is due to the formation of simpleton without departing from the main quantity in the last CVG, therefore, the accumulation of the virus occurs in CVG and inside cells. In a culture vessel with add 5-7 ml of 0.01 M Tris model HC1, pH=7.6 and the detergent Triton X-100 to a final concentration of 2-3%. The suspension is incubated for 1 h at (36±1)°C and stirring, is then treated with ultrasound 22 Hz 2 times in 20 seconds. The cell lysate is released from the cell detritus sedimentation QUE at 4-8°C for 8-12 h or low-speed centrifugation (rotor JA-14 centrifuge J2-21 ("Beckman, USA)at 10,000 rpm for 20 min at 4°C.

Example 2. Selection conditions chromatographic purification of the complex antigen

DEAE-pulp ("Whatman", Sweden) prepared according to the recommendations of the manufacturer. The column with DEAE-cellulose balance buffer 20 mm Tris-HCl, pH to 7.6. United su is pensio antigenic material three strains of measles virus to be applied on the column at a rate of 1 ml suspension of 1 ml of the sorbent. After application the column was washed with the same buffer. The elution of sorbed material is carried out by steps concentrations of NaCl in the same buffer solution to reduce the optical density at a wavelength of 280 nm in the output column with the solution to the "zero" values. Each faction examine the quality of the antigen sensitivity and specificity by ELISA (see table 3).

Example 3. Chromatographic purification of the complex antigen

The column with DEAE-cellulose balance buffer 20 mm Tris-HCl, pH to 7.6. Combined suspension antigenic material three strains of measles virus to be applied on the column at a rate of 1 ml suspension of 1 ml of the sorbent. After application the column was washed with the same buffer containing 0.2 M NaCl. The elution of the target complex antigen conduct 0.4 M NaCl in the same buffer. The target fraction is controlled to match the quality of antigen sensitivity and specificity by ELISA.

Table 3
The ELISA results in the formulation of positive and negative samples using immunosorbent made on the basis of different fractions of the chromatographic purification of complex antigen
The concentration of NaCl for elution fraction of the antigen with the number of the NCI OF positive sampleOF negative sampleOP(+)/OP(-)
Rezorbiruetsa fraction (slippage)0,1170,2560,5
0.1 M NaCl0,0750,1790,4
0.2 M NaCl0,1870,1201,6
0.3 M NaCl1,4590,12112,1
0.4 M NaCl2,4530,16316.2
0.5 M NaCl1,4690,2047,2
0.7 M NaCl0,5520,1922,9
1 M NaCl0,3400,1841,8

The output of high-purity complex antigen with 3 l KUR and lysate of cells is 15-20 mg

Example 4. Manufacturer immunosorbent assay

Immunosorbent prepared by the addition of 0.20±0.06 ml complex antigen measles virus after purification on column (see Example 3) 1,00±0,01 l R-RA sorption. The composition of R-RA sorption: 2,00±0.01 g of sodium carbonate, and 0.50±0.01 g of sodium azide, 0,400±0,012 ml of 2% phenolphthalein, purified water to 1 liter. Immunosorbent stirred on a magnetic stirrer for 1 h at a temperature of from 18°C to 24°C.

Deposition and sorption immunosorbent assay for tablets: for sorption use tablets Nunc Medisorp - "Nunc a/S, Denmark, or similar. Filling volume holes - 100-105 mm. Sorption was carried out at a temperature from 17°C to 27°C, time of sorption - 18-20 hours

Example 5. The method of use of a set of reagents for one-step ELISA Anti-measles Ig Resorbent"

For the analysis used the serum or plasma of human blood. For analysis only 10 μl of the sample. For the detection of antibodies of class G to the measles virus, you can use the serum (plasma) blood of the person as freshly prepared and not stored for more than 7 days at a temperature of from 2°C to 8°C in the absence of microbial contamination or not more than 12 months at a temperature of minus 20°C. freeze serum or plasma to a temperature of minus 20°C not more than two times.

For the reaction to all wells contribute 90 ál R-RA is svedeniya sample (composition: 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 hole in the tablet A1 contribute 10 ál K1+in wells B1 and C1 make 10 ál K2+. In well D1 contribute 10 ál To. In the remaining wells contribute 10 µl sample serum (final dilution of specimens and controls - 10 times). The solution is mixed by pipetting and incubated for 40 min at 37°C. after incubation the contents of the wells is removed and the plate washed seven times working solution FSB So In every hole making 100 ál of R-RA chromagen and the plate is placed for 15 min in the dark place at a temperature of 37°C. the Reaction is stopped by introducing into each well of the tablet in 50 µl of stop solution.

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

Psiv. calculated by the formula:

Psiv.=Opsys.×0,25: (APK+AVG)

where Opseu. - the OD value of the test serum.

Asyv. (the titer of antibodies in IU) R is schityvat by the formula:

Asyv.=10(Psiv.-a)÷b

where a and b are constants specific to each series specified in the Instructions that accompany each set).

An example of the calculation data obtained are given in table 4.

Table 4
An example of the calculation of the titer of antibodies to measles virus in the test serum
(APK+AVG) is the average OD value in the wells A1 and B12,919 - range Applications
(APK+AVG) is the average OD value in the wells C1 and D1(0,273+0,275):2=0,274 - range of Application
The lower boundary of the positive values>(APK+cf)=0,274
The upper boundary of the negative values<(APK+cf)-20%=0,220
The OCM - value OP in well E10,056<(APK+cf)-20%=0,220
The above values indicate the results of ELISA
Opsys. - the OD value of the test serum1,920
Psiv.=Opsys.×0,25: (APK+AVG),920×0,25:0,274=1,752
and - the value of the constant Application1,4
b - the value of the constant Application2,1
(Psiv. - a):b(1,752-1,4):2,1=0,168
Asyv.10has 0.168=1,47
Response: the specific activity of the serum 1,47 IU/ml

Example 6. Using a set of reagents "Anti-Measles Ig Resorbent" on the basis of the inventive antigen in clinical practice

1. During outbreaks of infectious diseases, Blagoveshchensk in 2010, a set of reagents "Anti-Measles Ig Resorbent" was used for serological confirmation of clinical diagnosis of measles". In 2 patients 2-3 days after the onset of clinical signs of disease and in 2-4 weeks spent drawing blood and obtaining sera for serological confirmation of the diagnosis of measles." Increasing titers of specific antibodies in paired sera studied in ELISA using commercial kits "Melisa Measles-IgM production CJSC "MBS" and in parallel with experimental series "Anti-Measles Ig Resorbent", the main component of which is immunosorbent on the basis of a complex antigen measles virus. The diagnosis of measles" confirmed the Ali after PCR with specific primers for C-terminal part of the gene encoding the NP protein of measles virus, and sequencing amplification of the fragment.

The set of "Melissa Measles-IgM revealed specific IgM antibodies to measles virus in both patients. When setting ELISA kit Anti-Measles Ig Resorbent" these patients also noted the presence of specific IgM antibodies to measles virus. When determining the rise in the titer of specific antibodies in paired sera with Anti-Measles Ig Resorbent" in both patients was zaregistrirovan at least 4-fold increase of specific IgG antibodies to measles virus.

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

Thus, the set of reagents "Anti-Measles Ig Resorbent" can be used for early detection of antibodies to measles virus, as well as for the detection of specific antibodies in paired sera obtained from patients, and thus to identify, remove or confirm the diagnosis in the presence of a disease with clinical manifestations characteristic of measles.

2. In the period from 2005 to 2010, in the Novosibirsk region was carried out to study the titre of specific antibodies to measles virus in the blood serum of children age 6 years prior to the second vaccination against measles. The titers of specific antibodies was studied in the reaction of rtga (by the conventional method using erythrocytes monkeys Macaca mulatta) and ELISA (using the proposed set of reagents "Anti-Measles Ig Resorbent").

The results of the comparison of the ELISA and rtga to determine measles antibodies in the serum of children showed that using "Anti-Measles Ig Resorbent" specific antibodies were identified 235 sera from 237 investigated sera. A similar result (235 sera from 237) were obtained using rcga. Simple averages of the specific activity of measles antibodies defined by a set of "Anti-Measles Ig Trearment were higher than specified in RTCA (6,2 IU/ml and 3.3 IU/ml, respectively).

Thus, the set of reagents for one-step ELISA Anti-Measles Ig Resorbent" can be used to determine the strength of individual immunity to the disease "measles".

1. Complex antigen measles virus used as a component of an enzyme immunoassay system for the diagnosis of antibodies to measles virus obtained from the culture fluid containing strains of measles virus Leningrad 16 with a titer of not less than 105.0TCD50/ml, Edmonston not less than 106.0TCD50/ml, NovO/96 - not less than 108.0TCD50/ml by inactivation of its infectious activity of the detergent and the release of protein from the cell lysate by chromatographic purification of at least 2 μg/ml with a purity of not less than 70%.

2. Complex antigen according to claim 1, characterized in that the culture of the other vaccinated fluid obtained during separate cultivation specified in claim 1 strains of measles virus in monolayer culture of Vero cells with subsequent mixing of cultural vaccinated liquids in the ratio 1:1:1 by volume.



 

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