[PHOSPHAN microplate technology-based microarray for the determination of IgG antibodies against West Nile and Crimean-Congo hemorrhagic fever viruses].

The paper demonstrates it possible to work out a phosphorescence analysis (PHOSPHAN) microplate technology-based microarray for concurrently examining human sera and detection of their specific IgG antibodies against two heterological West Nile and Crimean-Congo hemorrhagic fever viruses. The sensitivity and specificity of the microarray were comparable with those of enzyme immunoassay with separate sample testing. The advantages of PHOSPHAN were associated with the microplate format of an immunoassay and its enhanced multiplexity, which may contribute to the lower cost of clinical sample testing.

[Immunochip for differentiation of IgG to tick-borne encephalitis and West Nile fever viruses].

AIM: To demonstrate the possibility of development of test based on phosphorescent analysis (PHOSPHAN) for simultaneous identification and differentiation of specific IgG to tick-borne encephalitis (TBE) and West Nile fever (WNF) viruses. MATERIALS AND METHODS: Twenty six serum samples from patients with TBE, twenty five from WNF, and sixty six fromclinically healthy donors were used for the study. Immunologic analysis was performed in plate wells with active microzones "printed" on the wells' bottom and corresponding the complex of virus-specific antigens with immobilized monoclonal antibodies; internal control of specificity was included in each well. Species specificity of antibodies was determined on the basis of not less than 2-fold elevation of value of positivity coefficient (P/N) of sample studied with homologous antigen compared to heterologous one. RESULTS: PHOSPHAN provides simultaneous detection of IgG in human serum to two related flaviviruses: TBE and WNF viruses. Usage of P/N criterion assessed with homologous and heterologous antigen allowed correct determination of species specificity of antibodies in 90% of serum samples from patients with TBE and WNF CONCLUSION: PHOSPHAN allows to detect and differentiate IgG to TBE and WNF viruses during testing of one serum sample in one plate well without decrease of sensitivity compared to enzyme immunoassay with separated testing of samples on two viruses. This provides savings of biomaterial, which is an advantage compared to enzyme immunoassay.

[Diagnostic efficacy of phosphorescent immunochips for serologic diagnostics of tick-borne encephalitis].

AIM: To assess sensitivity and specificity of phosphorescent immunochips developed by the authors on the basis of microplate phosphorescent assay (PHOSPHAN) for detection of IgM and IgG antibodies to tick-borne encephalitis virus (TBEV) in sera of patients and to compare results of PHOSPHAN assay with results obtained by lanthanide immunofluorescence assay (LIFA) and solid-phase enzyme immunoassay (SPEIA). MATERIALS AND METHODS: Two hundred sixty one serum samples were tested, including 155 samples from 74 patients with clinical diagnosis of TBE confirmed by serologic identification of IgM antibodies to TBEV. Sera were collected in 2003 in Perm region from persons, which fell ill during seasonal increased activity of ticks-vectors of TBEV, as well as from healthy blood donors. Phosphorescent immunochip corresponds 96-well plate with 4 active microzones formed on the bottom of each well, which are able to detect specific IgM and IgG antibodies to TBEV. Immune reaction was visualized by conjugate of streptavidin with Pt-coproporphyrin. Intensity of fluorescence was measured by scanning the bottom of previously dried microwell with scanner IFI-02. RESULTS: Comparable sensitivity and specificity of POSHPHAN assay, LIFA and SPEIA was demonstrated for detection of IgM and IgG antibodies to TBEV in samples. Immunoluminescence-based PHOSPHAN assay and LIFA were more sensitive for analysis of sera with low titer of specific IgM antibodies. CONCLUSION: PHOSPHAN assay could be used for early serologic diagnostics of TBE as well as for assessment of antibody level for control of efficacy of treatment in patients with prolonged illness or level of protective immunity in vaccinees.

[Testing for antibodies to tick-borne encephalitis virus using blood dried on filter paper].

Possibility to use blood dried on filter paper for serological testing on antibodies to tick-borne encephalitis (TBE). Sensitivity and specificity of specific IgM detection in dry stains of blood by lanthanide fluorescence immunoassay was 94.9% (86.9-100%) and 97.5% (94-100%) respectively, compared with results obtained in tests of sera. Agreement in positive and negative results of tests for IgM against TBE in 562 serum samples and dry blood stains was 95.3%. During analysis of both types of biomaterial high degree of correlation was observed between intensity of fluorescence when testing for both IgM (r=0.86700; p=0.05; n=562), and IgG (r=0.83883; p=0.05; n=337) toTBE virus. Use of this mildly invasive technique of blood draw is reasonable during conduction of large-scale population studies for seroepidemiologic monitoring, investigation of disease outbreaks, control of effectiveness of vaccination against TBE, assessing of level of specific immunity in population of endemic regions, control of treatment, and serologic diagnostics of acute TBE in hospitalized patients, in which blood draw is difficult to perform.

[Phosphorescent microanalysis as a new technical platform for molecular diagnostics].

The authors developed a method of phosphorescent multiplex microanalysis (PHOSPHAN) as a new technological platform for a wide scope of molecular diagnostic tasks, and consider the prospects of its application in the article. PHOSPHAN combines the potential of solid-phase microplate analysis with the principle of microarray laser scanning of microzonules with biospecifically bound analyte on the surface of the bottom of microplate holes with consequent real-time registration of the phosphorescent signal. The sensitivity of the instrumental detection system is approximately 1000 molecules of Pt coproporphyrin mark in the illuminated area of scanning of 30 microns in diameter. PHOSPHAN makes it possible to detect separate microbial cells in samples and increases the sensitivity of analyte detection in microaliquots eluted from blood spots dried on paper blanks. All the key elements of this technology are protected with Russian patents.

[Lanthanide immunofluorescence assay for the serodiagnosis of tick-borne encephalitis].

Lanthanide immunofluorescence assay (LIFA) was used to detect IgM of antibodies to tick-borne encephalitis (TBE) virus in 791 patients who had been fallen ill with acute febrile diseases in the period of seasonal activity of carrier ticks in the endemic region of Russia (the Perm Region) (1786 sera being tested). This assay was equally effective as the commercial enzyme immunoassay test system (EITS) in the early serological diagnosis of TBE, verifying the clinical diagnosis in about 70% of patients just within the first week of disease. At the same time, the sensitivity of LIFA was much (nearly 5 times) higher than that of EITS in revealing antibody IgM in patients with chronic TBE, as well as in those with the acute course of disease, accompanied by the low level and untypical trend of accumulation of antibodies ofthis class.

[Choosing the threshold level in the detecting of antibodies to the causative agents of Ixodes tick-borne borreliosis by lanthanide immonofluorescence assay].

The paper describes the algorithm of choice of the threshold level of fluorescence, which identifies the positive and negative results of lanthanide immunofluorescence assay when IgM and IgG antibodies to the causative agents of Ixodes tick-borne borreliosis are detected in the sera of patients. The diagnostic specificity index of the method in detecting antibodies of both types was 95.3% for the chosen value. According to the time of sampling, the diagnostic sensitivity indices for IgM and IgG antibodies were 10.4 to 40.9% and 3.3 to 18.2%, respectively.

[Designing and clinical testing of immune-enzyme and immunofluorescence test systems for serodiagnosis of ixodes borreliosis]. / Razrabotka i klinicheskaia aprobatsiia immunofermentnykh i immunoliuminestsentnykh test-sistem dlia serodiagnostiki iksodovykh kleshchevykh borreliozov.

The methods of immune enzyme assay (MIEA) and of lanthanide immunofluorescence analysis (LIFA) were used to work out the test systems for the detection (in blood serum of patients) of specific IgM IgG antibodies to the B. burgdorferi spirochete--a causative agent of ixodic borrelioses. The test system was clinically tested versus the indirect immunofluorescence reaction (IIFR) and commercial immune enzyme test system (CIET). The results of antibodies' detection were shown to correlate with the analysis data for the same sera in IIFR and to be in line with a real presence or absence of the disease. Test systems based on LIFA were proven to be most sensitive and specific.

[Membrane-filtration immunoassay: reagents, methods and the diagnostic and technical means for detection]. / Membranno-fil'tratsionnyi immunoanaliz: reagenty, metody, diagnosticheskie i tekhnicheskie sredstva indikatsii.

The comprehensive development of dot-EIA made at the State Research Institute of Biological Instrument-Making Industry has provided devices KIMF-02 and KIMF-03), a base of chemical reagents, immunoassays, test systems for detection of a wide range of causative agents of viral and bacterial infections, that of serodiagnosis of their related diseases. The KIMF-02 kit has undergone engineering and medical tests and recommended for the Ministry of Health of the Russian Federation to produce them in stock. The kit includes all required for analysis even in an ill-equipped laboratory, a set of attached agents ensures a valid visual recording of results. The developed procedures and test systems allow the immunoassay to be as sensitive as TIFA; however, they are laborious and much simpler in design. The simple and rapid procedures of dot-EIA are recommended for incorporation into the a package of laboratory methods for verification of the accumulation of virus-specific antigens in various biological substrata, environmental samples, for control of the activity of antigens and antibodies used in serological tests, for detection of specific antigens in the clinical samples, and for serodiagnosis of infections.

[Diagnosis of the active form of cytomegalovirus infection based on determination of IgG antibodies to the immediate-early cytomegalovirus protein by lanthanide immunofluorescence analysis]. / Diagnostika aktivnoi formy tsitomegalovirusnoi infektsii na osnove opredeleniia IgG-antitel k predrannemu belku tsitomegalovirusa metodom lantanidnogo immunofliuorestsentnogo analiza.

A system for time-resolved fluoroimmunoassay (TR-FIA) is developed for detecting early IgG during the active stage of cytomegalovirus infection on the basis of CMV-control enzyme immunoassay system. The antigen is cloned and expressed in E. coli recombinant main immediate early protein of human CMV. The active form of CMV infection was detected additionally in 5 out of 25 women who gave birth to sick babies by means of the new test system in complex with other laboratory diagnostic methods. The test is recommended for prenatal diagnosis of active CMV infection in pregnant women and for predicting the risk of neonatal disease.

[The development and testing of test systems for the determination of herpes simplex virus and cytomegalovirus antigens by lanthanide immunofluorescence analysis]. / Razrabotka i aprobatsiia test-sistem dlia opredeleniia antigenov virusa prostogo gerpesa i tsitomegalovirusa metodom lantanidnogo immunofliuorestsentnogo analiza.

On the basis of the lanthanide immunofluorescent assay (LIFA) test systems for the determination of herpes simplex virus (HSV-LIFA) and cytomegalovirus (CMV-LIFA) antigens have been developed. The test system HSV-LIFA includes the sandwich of monoclonal antibodies (McAb) HSV A.3.3. or B-11 to type 2 HSV, strain BH; the test system CMV-LIFA includes the sandwich of rabbit McAb to CMV, strain AD 169. The approbation of the test systems has revealed that they insure the specific detection of HSV and CMV antigens in clinical specimens (urine, blood, liquor, saliva), the LIFA results well correlating with the data on the isolation of viruses in cell cultures, with the results obtained by other diagnostic methods and with clinical manifestations of diseases. LIFA has been shown to be more sensitive than the enzyme immunoassay.

[Indication and identification of California serogroup viruses in experimentally infected Ae. aegypti mosquitos by a lanthanum immunofluorescent analysis method]. / Indikatsiia i identifikatsiia virusov kaliforniiskoi serogruppy v éksperimental'no zarazhennykh komarakh Ae. aegypty metodom lantanidnogo immunofliuorestsentnogo analiza.

The sensitivity and specificity of lanthanide fluoroimmunoassay (LFIA) and enzyme immunoassay (EIA) for indication and identification of California antigens in suspensions of infected Aedes aegypti mosquitoes are compared. The sensitivity of LFIA was 2.3 Ig TCD50/ml, EIA was less sensitive and detected virus antigens in mosquito suspensions at infective titer of 4.1 Ig TCD50/ml. If a pool contained mosquitoes infected with different California viruses, they could be identified only at the level of serological complex.

[Further study of hantavirus circulation in the Russian Federation]. / Dal'neishee izuchenie tsirkuliatsii khantavirusov v Rossiiskoi Federatsii.

Lung specimens of 1514 small mammals of 35 species captured in 1991-1995 at 9 territories of Russia were tested in ELISA for virus antigens of hemorrhagic fever with the renal syndrome (HFRS). The antigens were detected in lung specimens of Clethrionomys glareolus, Microtus arvalis, Microtus gregalis, Microtus fortis, Arvicola terrestris, Apodemus agrarius, Micromys minutus, and Sorex sp., well known as Hantavirus reservoirs, captured in the Vologda, Yaroslavl, Saratov, Astrakhan, and Chita regions. Infection of Microtus maximoviczii revealed in the Chita region was first found in China. Previously there were no reports about the circulation of hantaviruses in this region. Our study added one more host to the list of HFRS virus hosts: Meliones tamariscinus.

[Comparative evaluation of test-systems for detection of Venezuelan equine encephalomyelitis virus and variola virus using dot immunoenzyme assay and solid-phase lanthanide immunofluorescent assay]. / Sravnitel'naia otsenka test-sistem dlia opredeleniia virusov venesuél'skogo éntsefalomielita loshadei i ospovaktsiny metodami tochechnogo immunofermentnogo analiza i tverdofaznogo lantanidnogo immunofliuorestsentnogo analiza.

The sensitivity and specificity of 4 experimental test systems for dot enzyme immunoassay (dot-EIA) and solid-phase lanthanide immunofluorescent analysis (SP LIFA) were studied with Venezuelan equine encephalomyelitis (VEE) and variolovaccinia viruses. Test systems for SP LIFA proved to be 25 times more sensitive than those for dot-EIA. The test systems were highly specific and did not react with the heterologous viruses and proteins. Diagnostic agent for detecting VEE virus in dot-EIA was false-positive with Sindbis virus in low dilutions. The first trials of the Russian test systems for dot-EIA and SP LIFA showed that these systems rapidly and reliably detect VEE and variolovaccinia viruses in liquid samples.

[Use of 3,3',5,5'-tetramethylbenzidine as substrate in immunoenzyme analysis]. / Ispol'zovanie 3,3',5,5'-tetrametilbenzidina v kachestve substrata pri immunofermentnom analize.

The possibility of using 3,3",5,5'-tetramethylbenzidine and its derivatives in solid-phase enzyme immunoassay and in dot enzyme immunoassay on membrane filters has been demonstrated in experiments with arboviruses. All the tested substrates were fit for specific indication of viral antigens and provided an adequate sensitivity of analyses in comparison with the routinely used substrates, ortho-phenylenediamine and ortho-toluidine. An important advantage of the tested substrates is the absence of carcinogenicity.

[The detection of antibodies to the Venezuelan equine encephalomyelitis virus by immunoenzyme analysis using antigen purified in a water-polymer system]. / Vyiavlenie antitel k virusu venesuél'skogo éntsefalomielita loshadei metodom immunofermentnogo analiza s primeneniem ochishchennogo v vodno-polimernoi sisteme antigena.

Human and animal sera were tested for the presence of antibodies to Venezuelan equine encephalomyelitis (VEE) virus by direct enzyme immunoassay (EIA). For the test, the plates were sensitized with a VEE virus preparation purified in a two-phase system of water-soluble polymers. The proposed EIA variant was as specific as that with VEE antigen obtained by fractionation on sucrose cushion, but more sensitive. The high specificity of the assay allowed the antigen purified in the water-polymer system to be used for investigation of antigen relationships among viruses of the VEE complex.

[The demonstration of the Venezuelan equine encephalomyelitis virus by the method of indirect lanthanide immunofluorescent analysis]. / Indikatsiia virusa venesuél'skogo éntsefalomielita loshadei metodom nepriamogo lantanidnogo immunofliuorestsentnogo analiza.

A test system of indirect time-resolved fluoroimmunoassay (TR-FIA) was developed and tested on an alpha-virus, Venezuelan equine encephalomyelitis virus. The indirect TR-FIA test system was shown to be highly effective in the detection of antigens of this virus. Not differing in specificity from the direct TR-FIA, the newly developed test system was 4 times as sensitive.

[The design and trial of conjugates for performing lanthanide immunofluorescence analysis]. / Konstruirovanie i aprobatsiia kon''iugatov pri postanovke lantanidnogo immunofliuorestsentnogo analiza.

The possibility of using a number of complexons for labeling of antibodies to Venezuelan equine encephalomyelitis virus and to adenovirus with europium ions was studied. The resultant conjugates, irrespective of the type of complexon, were shown to retain their immunochemical activity and could be used for lanthanide immunofluorescence analysis of virus-specific antigens.

[The use of sectional polystyrene plates in the set-up of lanthanide immunofluorescence analysis]. / Ispol'zovanie razbornykh polistirolovykh planshetov pri postanovke lantanidnogo immunofliuorstsentnogo analiza.

The authors have examined the possibility of using sectional polystyrene plates, made in this country, in time-resolved fluoroimmunoassay (tr-FIA) with Venezuelan equine encephalomyelitis and tick-borne encephalitis arboviruses, and with influenza A virus. The plates presensitized with specific antibodies were found fit for the detection of the antigens of the above viruses. These plates are not recommended for the detection of influenza A virus-specific proteins adsorbed directly onto the microplate surface.