Ifa system test vector best. Instruments and reagent kits for ELISA produced in Russia Kundelsky R.V., Ph.D.
Leading Russian manufacturers in the segment of reagent kits for the diagnosis of infections: - Vector-Best CJSC, Novosibirsk; - NPO Diagnostic Systems LLC, Nizhny Novgorod; - JSC "Ekolab", Elektrogorsk MO. In the field of reagent kits for the diagnosis of non-infectious diseases and physiological conditions: - Alkor-Bio LLC, St. Petersburg; - Vector-Best CJSC, Novosibirsk; - Hema-Medica LLC, Moscow. In the segment of reagents for laboratory diagnosis of allergies: - Alkor-Bio LLC, St. Petersburg; - Vector-Best CJSC, Novosibirsk; - NPO Immunotex LLC, Stavropol. Production of laboratory equipment: - Picon LLC, Moscow
Assessment of the degree of possible import substitution based on the range of tests Nomenclature of analytes, units. Approved for use in the Russian Federation Produced in the Russian FederationDegree of possible import substitution Markers of infectious diseases % Markers of non-communicable diseases and indicators of physiological conditions % Allergy markers % Total: %
Assessment of the degree of import substitution based on the market volume of products for tablet ELISA Market segmentsMarket volume in the Russian Federation, million rubles. Sales volume of domestic manufacturers, million rubles. Share of import substitution Markers of infectious diseases % Markers of non-communicable diseases and indicators of physiological conditions % Allergy markers % Equipment50051% Total: %
Assessment of the degree of import substitution based on the volume of the market for products for immunochemical methods in general Market segmentsMarket volume in the Russian Federation, million rubles. Sales volume of domestic manufacturers, million rubles. Share of import substitution Markers of infectious diseases % Markers of non-communicable diseases and indicators of physiological conditions % Allergy markers % Equipment % Total: %
Problems of domestic manufacturers in the industry: The ELISA method is becoming outdated. There is a gradual replacement of domestic IF reagents with imported chemiluminescent ones; The existing public procurement system stimulates either the purchase of the cheapest goods or imported products with unique characteristics; One of the main reasons for the insufficient development of domestic manufacturers of automatic equipment and localization of production of imported products is the customs administrative barrier; The main problem of recent times is the collapse of the system of state registration of new products in the Republic of Belarus. The admission of new products to the market has practically stopped and those foreign manufacturers who previously registered long lists continue to have an advantage over domestic ones; The main reason for the technological lag is the inability to obtain the right to produce products compatible with modern equipment; The low degree of consolidation of domestic manufacturers and constant price wars among themselves do not make it possible to develop advanced products and compete with leading players on equal terms, including in foreign markets.
Proposals: Within the FCC, introduce a non-competitive procedure for public procurement of medical products with subsequent position-by-item publication. Determining financing limits based on the average market price will give certain advantages to domestic producers, will limit the consumption of expensive imported reagents and control the use of budget funds more efficiently; Promote the consolidation and consolidation of domestic manufacturers in order to concentrate resources on the development of modern products and enter the international market. The non-competitive procedure for government procurement will quickly leave only truly competitive enterprises on the market; Allocate government funds to stimulate, on the principles of public-private partnership, the development of a range of tests sufficient to ensure the biological safety of the country; Customs benefits should be for components, not for finished products; Harmonize the registration system with the European one, where medical devices with a low level of potential risk are registered on the basis of a declaration of conformity.
DRUGS
EVALUATION OF NEW ELISA TEST SYSTEM
"Rotavirus-antigen-ELISA-BEST"
12Zhirakovskaya E.V., 3Ignatiev G.M., 3Indikova I.N., 12Tikunova N.V.
1 Federal State Budgetary Institution State Scientific Center VB "Vector" of Rospotrebnadzor, Koltsovo village, Novosibirsk region;
2 Institute of Chemical Biology and Fundamental Medicine, Novosibirsk;
3 State Institute for Standardization and Control of Medical Biological Preparations named after. L.A. Tarasevichag Moscow
The results of tests of sensitivity, specificity and reproducibility of a new set of reagents “Rotavirus-antigen-ELISA-BEST” developed at JSC “Vector-Best” (Novosibirsk) are presented. The data obtained allow us to predict the diagnostic reliability of the results when using this test system to detect group A rotavirus antigen in clinical material.
Key words: ELISA test system, efficiency, rotavirus A
Group A rotaviruses (family Reoviridae, genus Rotavirus) are the most common cause of severe gastroenteritis in young children worldwide. Adults with weakened immune systems often also become ill. Rotavirus infection (RVI) is a highly contagious disease with multiple routes of spread. The source of infection is a person with a manifest or asymptomatic form of the disease, as well as a virus carrier. Among children and adults, RVI can manifest itself in the form of sporadic cases, local group diseases, outbreaks and is widespread. The fecal-oral mechanism of transmission of this infection is realized through food (milk and dairy products, baby food), water and contact-household routes.
Making a diagnosis of rotavirus gastroenteritis based on the clinical picture, especially with sporadic incidence, is a certain difficulty, since the symptoms characteristic of this infection differ little from the symptoms of other acute intestinal infections (AEI) of various etiologies. Differential diagnosis in patients with rotavirus gastroenteritis is carried out both with foodborne toxic infections and with other viral infections (noroviruses, astroviruses, adenoviruses, coronaviruses, Coxsackie and ECHO enteroviruses) and bacterial (salmonellosis, dysentery, cholera, yersiniosis, opportunistic microorganisms) etiology. Unfortunately, not all specialized medical institutions in the Russian Federation carry out diagnostics of rotavirus infection.
Diagnostic methods for RVI are aimed at detecting whole virions, viral antigen or virus-specific RNA in feces. A promising approach to the direct detection of viruses both in clinical material and in environmental objects is the reverse transcription-polymerase chain reaction (RT-PCR) method. In recent years, test systems have been created based on modern scientific developments with a combination of various methods for detecting rotaviruses: multiplex PCR with hybridization-fluorescence detection of amplification products “by end point”; endpoint immuno-PCR (IPCR) with real-time detection; quantitative RT-PCR with real-time detection. Research laboratories use electron microscopy to quickly identify rotaviruses. However, all of the above methods are quite labor-intensive and require expensive equipment and highly qualified personnel. Therefore, when conducting laboratory diagnostics in hospitals and outpatient settings, preference is given to methods based on the detection of viral antigen in feces using enzyme-linked immunosorbent assay (ELISA) with mono- and polyponal antibodies to rotaviruses. This method is available for practical laboratories, is easy to set up and allows you to quickly obtain results.
The purpose of this work is to study the diagnostic effectiveness of the new set of reagents “Rotavirus-antigen-ELISA-BEST” developed at JSC “Vector-Best”, Novosibirsk.
^September-December
Materials and methods
The study material included fecal samples from young children diagnosed with acute intestinal infection and without clinical manifestations of intestinal infection, who were undergoing inpatient treatment in the departments of intestinal infections and respiratory infections of the Children's City Clinical Hospital No. 3 of Novosibirsk. Fecal samples were collected in disposable sterile plastic containers in a volume of 2-3 ml upon patient admission to the hospital department and stored at -20 °C for 15 days. Longer storage of the material was carried out at - 70 °C.
A panel of 104 fecal samples, previously tested for the presence of ACI pathogens, was used in this work. Of these:
30 samples in which only group A rotaviruses were detected by ELISA and RT-PCR; genotyping using the RT-PCR method showed that fecal samples contained rotaviruses of genotype PG1 (18 samples), PG2 (4 samples), PG3 (3 samples), PG4 (2 samples), PG4 (1 sample), PG9 (1 sample) , PG3 (1 sample);
14 samples in which only noroviruses of the second genotype were identified by RT-PCR, which was confirmed by determining the nucleotide sequence of the 5" region of the capsid gene located on the norovirus genome in the region 5085-5485 h.;
15 samples in which only astroviruses were identified by RT-PCR, which was confirmed by determining the nucleotide sequence of the 5" region of the capsid gene located on the astrovirus genome in the region 4526 - 4955 nt; 15 samples in which only adenoviruses were identified by PCR ;
30 fecal samples (control) from children without clinical manifestations of intestinal infection hospitalized in the respiratory department of the hospital; Preliminary analysis did not identify the above-mentioned viral pathogens in these samples.
Testing of samples for the presence or absence of rota-, noro-, astro- and adenoviruses was carried out using the RT-PCR method using commercial kits registered in the Russian Federation “AmpliSens No. virus 1, 2 genotypes - 306/322”, “AmpliSens Astrovirus -165”, “AmpliSens Adenovirus - 462”, “AmpliSens Rotavirus - 290” (produced by the Central Research Institute of Epidemiology, Russian Federation). The samples were examined in accordance with the instructions for use of the corresponding reagent kits during the expiration date. Rotavirus-positive samples were genotyped using RT-PCR. Determination of the presence or absence of rotavirus antigen was also carried out by ELISA using the commercial IDEIA™ Rotavirus test system (DakoCytomation, UK) in accordance with the instructions for the kit.
When testing the “Rota-virus-antigen-ELISA-BEST” reagent kit, all of the above samples (104) were encrypted and tested three times with the test kit. After completion of the tests, the samples were decrypted and the results were analyzed.
Results and discussion
The sensitivity of the Rotavirus-antigen-ELISA-BEST reagent kit was assessed by the number of coincidences of positive results (in%) testing samples using the tested test system and reference drugs. At the same time, it was revealed that when using the “Rotavirus-antigen-ELISA-BEST” reagent kit, all 30 samples with the confirmed presence of rotavirus A were positive (Table 1). Consequently, the sensitivity of the test set of reagents in detecting rotaviruses A was 100%. It should be noted that the panel included samples with seven different genotypes of rotaviruses, and all of them were successfully detected by the “Rota virus rus - antigen - AND FA-B EST” reagent kit.
The specificity of the test system was assessed by the number of coincidences of negative results (in %) of testing samples using the tested test system with those passed preliminary studies. To assess the specificity, the panel used included 30 samples in which viral pathogens were not identified, as well as 14 samples in which only noroviruses of the second genotype were detected using the RT-PCR method, 15 samples in which the RT-PCR method detected only astroviruses; 15 samples in which only adenoviruses were detected by PCR. It was determined that in 69 of 74 negative samples, the optical density values when detected by the Rotavirus-Antigen-ELISA-BEST reagent kit for the presence of rotavirus A did not exceed the background values, that is, the values obtained as a result of measuring the optical density in control negative samples. Two samples containing astroviruses, one sample containing a norovirus of the second genotype, one sample containing an adenovirus, and two samples in which none of the above viral pathogens were detected demonstrated positive optical density readings. It should be noted that in samples in which none of the viral pathogens had previously been detected, positive signals were recorded in only one of the repeated studies (Table 2). Thus, the specificity of the Rotavirus-antigen-ELISA-BEST reagent kit was 93.2%.
During the tests, the reproducibility of the results obtained when using the test set of reagents “Rotavirus-antigen-ELISA-BEST” on clinical material was assessed - all samples were tested three times in different experiments to determine the variation in results. In almost all cases, similar results were obtained: the test system detected all negative and positive samples equally. The exceptions were two samples in which none of the viral pathogens had previously been identified: in one of the replicates for both samples, the optical density values exceeded the OPcrit. It should be noted that the excess was insignificant (Table 2). Thus, the reproducibility of the results was 98.8%.
The results of the tests showed that the tested set of reagents “Rotavirus-antigen-ELISA-BEST” in terms of its diagnostic effectiveness - specificity, sensitivity and reproducibility - is comparable to those available in the Russian Federation for diagnostics.
chemical test systems, which makes it possible to predict the diagnostic reliability of the results when using the studied set of reagents “Ro-tavirus-antigen-ELISA-BEST” to detect the group A rotavirus antigen in clinical material.
Literature
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2. Vasiliev B.Ya., Vasilyeva R.I., Lobzin Yu.V.// “Acute intestinal diseases. Rotaviruses and rotavirus infection.” - St. Petersburg, Lan. - 2000.
3. Zhirakovskaya E.V., Tikunov A.Yu., Bodnev O.A., et al.// “BIOpreparations”. - 2008 - No. 2. - p. 15 - 18.
4. Zhirakovskaya E.V., Maleev V.V., Bodnev A.S. and others // JMEI - 2008 - No. 4. - With. 12 - 16.
5. Ignatyuk T.E., Golutvin I.A., Nasikan N.S., et al. // “Questions of Virology”. - 2003. - t. 48. - No. 6. - p. 1721.
6. Novikova N.A., Fedorova O.F., Epifanova N.V., Chup-rova A.B. // “Questions of Virology”. - 2007. - t.52. -No. 3 - s. 19-23.
7. Podkolzin A.T., Mukhina A.A., Shipulin G.A., et al.//
"Infectious diseases". - 2004. - vol. 2. - No. 4. - p. 85 -91.
8. Podkolzin A.T., Fenske E.B., Abramycheva N.Yu., et al.// “Therapeutic Archive”. - 2007. - t. 79. - No. 11. - p. 10-16.
9. Sergevnin V.I., Voldshmidt N.B., Sarmometov E.V., et al. // “Epidemiology and infectious diseases”. -2004.-No. 6.-p. 17-20.
10. Sergevnin V.I., Voldshmidt N.B., Sarmometov E.V., et al. // Hygiene and Sanitation. - 2007. - No. 1. - p. 56 -58.
11. Arcangeletti M. S., De Conto E., Pinardi F., at al. // Acta Biomed. Ateneo. Parmense. - 2005. -V. 76(3). - P. 165 -170.
12.Gladstone B.P., Iturriza-Gomara M., Ramani S., at al. // Epidemiol. Infect. - 2008. V. 136(3). - P. 399-405.
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14. Santos N., Honma S., Timenetsky Mdo C., at al. //J. Clin. Microbiol. - 2008. V. 46 (2). - P. 462 - 469.
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Evaluation of the sensitivity and specificity of the “Rotavirus-antigen-ELISA-BEST” reagent kit
Table 1
No. Number of samples Detection results with reference drugs IDEIA Rotavirus Astro-PCR Hopo 2-PCR Adeno-PCR Detection results with the “Rotavirus-antigen-ELISA-BEST” kit
1. 30 30 0 0 0 30
2. 15 0 15 0 0 2
3. 14 0 0 14 0 1
4. 15 0 0 0 15 1
Assessment of the specificity of the reagent kit “Rotavirus-antigen-ELISA-BEST>-
Table 2
PCR AmpliSense
rotavirus rotavirus rotavirus rotavirus
0,443 1,306 0,676 0,418
Rotavirus-antigen-ELISA-BEST JSC "Vector-Best"
OPCrit OPCrit OPCrit
0,250 0,263 0,251
> 4,000 > 4,000 > 4,000 3,926 > 4,000 3,939
> 4,000 > 4,000 > 4,000
> 4,000 > 4,000 > 4,000
£September
December 2009
AmpliSense
rotavirus rotavirus rotavirus rotavirus rotavirus rotavirus rotavirus rotavirus rotavirus rotavirus rotavirus rotavirus rotavirus rotavirus rotavirus rotavirus rotavirus rotavirus rotavirus rotavirus rotavirus rotavirus rotavirus rotavirus rotavirus rotavirus astrovirus astrovirus astrovirus astrovirus astrovirus astrovirus astrovirus
IDEIA Rotavirus DakoCytomation OPCrit = 0.150
0,401 0,322 1,659 0,566 0,518 1,278 1,285 0,809 1,160 0,407 0,218 0,703 1,889 1,069 1,302 0,879 1,842 0,747 0,793 1,013 1,124 0,670 0,726 0,683 0,814 0,997 0,206 0,052 0,050 0,034 0,040 0,048 0,040
Rotavirus-
OPCrit 0.250
> 4,000 3,933 3,987 3,918 3,853 3,972
> 4,000 3,864 3,879 3,897 3,800
> 4,000 3,981
> 4,000 3,713
> 4,000 4.000
> 4,000 3,989
> 4,000 3,872 0,088 0,103 0,230 0,240 0,268 1,819 0,062
antigen-ELISA-BEST "Vector-Best"
OPCrit OPCrit 0.263 0.251
> 4,000 > 4,000
> 4,000 > 4,000
> 4,000 > 4,000 3,821 3,899
> 4,000 3,964 3,845 3,923 3,962 3,871
> 4,000 3,929
> 4,000 3,881 3,884 > 4,000
> 4,000 > 4,000 3,800 3,851 3,818 >4,000
> 4,000 > 4,000
> 4,000 3,995
> 4,000 > 4,000
> 4,000 > 4,000
> 4,000 > 4,000 3,837 3,839
> 4,000 > 4,000
> 4,000 > 4,000
> 4,000 3,986
> 4,000 > 4,000
> 4,000 3,998
> 4,000 > 4,000
> 4,000 3,823 0,063 0,073 0,054 0,061 0,255 0,250 0,256 0,244 0,278 0,560 1,117 1,235 0,052 0,052
astrovirus astrovirus astrovirus astrovirus astrovirus astrovirus astrovirus astrovirus norovirus norovirus norovirus norovirus norovirus norovirus norovirus norovirus norovirus norovirus norovirus norovirus norovirus norovirus adenovirus adenovirus adenovirus adenovirus adenovirus adenovirus adenovirus adenovirus adenovirus adenovirus adenovirus
IDEIA Rotavirus DakoCytomation OIIKpHT = 0.150
0,043 0,043 0,041 0,052 0,040 0,046 0,041 0,040 0,037 0,039 0,030 0,045 0,032 0,030 0,037 0,042 0,034 0,039 0,043 0,043 0,045 0,039 0,050 0,034 0,050 0,043 0,050 0,042 0,041 0,042 0,038 0,047 0,039
PoTaBHpyc-aHTHreH-HOA-EECT 3AO “BeKTop-EecT”
OnKpHT OnKpHT OIIKpHT
0,250 0,263 0,251
0,115 0,075 0,082
0,053 0,046 0,058
0,233 0,198 0,189
0,144 0,105 0,128
0,243 0,062 0,073
0,043 0,040 0,046
0,069 0,043 0,041
0,143 0,044 0,058
0,220 0,206 0,230
3,475 2,577 2,405
0,223 0,247 0,240
0,232 0,236 0,231
0,048 0,042 0,041
0,121 0,085 0,093
0,132 0,111 0,174
0,122 0,052 0,063
0,061 0,044 0,054
0,073 0,035 0,048
0,089 0,046 0,046
0,047 0,043 0,044
0,041 0,039 0,044
0,083 0,046 0,038
0,168 0,074 0,097
0,247 0,118 0,099
0,248 0,251 0,242
0,243 0,259 0,250
0,054 0,048 0,055
0,048 0,040 0,037
0,058 0,045 0,046
0,053 0,045 0,049
0,069 0,058 0,065
0,912 0,344 0,379
0,089 0,037 0,042
December 2009
PCR AmpliSense
adenovirus adenovirus adenovirus adenovirus negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative
IDEIA Rotavirus DakoCytomation OPCrit = 0.150
0,040 0,044 0,039 0,041 0,260 0,044 0,046 0,042 0,047 0,041 0,039 0,048 0,055 0,035 0,039 0,040 0,041 0,033 0,046 0,049 0,048 0,038 0,039 0,029 0,037 0,036 0,043 0,043 0,039 0,042 0,034 0,037 0,041 0,036
Rotavirus - antigen - ELISA-BEST JSC "Vector-Best"
OPCrit O P Crit OPCrit
0,250 0,263 0,251
0,105 0,042 0,043
0,046 0,143 0,133
0,227 0,045 0,042
0,065 0,068 0,054
0,125 0,039 0,042
0,196 0,191 0,182
0,209 0,170 0,154
0,316 0,071 0,073
0,058 0,043 0,049
0,171 0,056 0,061
0,049 0,060 0,064
0,047 0,062 0,065
0,058 0,047 0,066
0,073 0,072 0,066
0,188 0,120 0,109
0,180 0,074 0,072
0,057 0,063 0,058
0,047 0,060 0,047
0,162 0,146 0,143
0,248 0,248 0,260
0,063 0,066 0,104
0,247 0,223 0,251
0,054 0,053 0,070
0,242 0,240 0,248
0,073 0,061 0,103
0,066 0,065 0,064
0,108 0,123 0,192
0,072 0,067 0,071
0,079 0,079 0,087
0,104 0,079 0,164
0,169 0,150 0,162
0,140 0,166 0,146
0,114 0,133 0,131
Number of definitions 96 (48 in duplicates)
Working tablet format: striped 12x8, broken into 1 hole.
Sensitivity: 1.5 U/ml.
Measuring range: 0-400 U/ml.
The volume of the test sample is no more than 25 µl.
Standardization of conditions for carrying out an enzymatic reaction with chromogen in a thermostatic shaker at 37ºC.
Set contents:
1. Striped plate 12 x 8 wells, ready for use - 1 pc.
2. Calibration samples ready for use (0-400 U/ml) colored with varying degrees of intensity depending on the concentration - 6 bottles.
3. Control sample - 1 bottle.
4. One-component conjugate, ready for use, not requiring dilution - 1 bottle.
5. Solution for diluting serums - 1 bottle.
6. Chromogenic substrate, one-component - tetramethylbesidine plus (TMB+) solution, ready for use, not requiring dilution - 1 bottle.
7. Phosphate-buffered saline solution with Tween - 2 bottles.
8. Stop reagent, ready for use - 1 bottle.
9. Film for sealing the tablet - 1 pc.
10. Stencil for constructing a calibration graph - 1 pc.
11. Reagent bath - 2 pcs.
12. Pipette tips for 5-200 µl - 16 pcs.
The tablet is packaged in a teak ziplock bag.
The stability of the FST-T working solution is at least 5 days at a temperature of +2...8°C.
Store the kit at a temperature of +2...8°C. Shelf life - 1 year from the date of production.
Registered with Roszdravnadzor.
