[The multiplex analysis of drug medicinals on the basis of technology of immunochips Phosphan.]

The new technique of multiplex qualitative analysis of narcotic, psychotropic remedies is developed on the basis of technology Phosphan using immunochips in the format of standard 96-wells plates, monoclonal antibodies to narcotic compounds and Pt-coproporphyrin as a long luminescent marker. The multiplex analysis was implemented using 20 mkl of human biological fluid (urine, blood serum or saliva) of 2 discs of 3.2 mm in diameter made of dried urine spot on paper. No preliminary processing or dilution of analyzed sample is required. The large range of measured concentrations was demonstrated under high sensitivity of analysis: 1 ng/ml of morphine and methadone, 0.5 ng/ml of barbiturates, 2 ng/ml of benzoylecgonine, methamphetamine, cannabinoids and benzodiazepines, 8 ng/ml amphetamine at variability of results no more than 15%. The approbation of technique was implemented using valid samples of urine (n=197) and blood serum (n=98) demonstrated that the technique permits to detect properly opiates, cocaine, cannabinoids, methadone, benzodiazepine, barbiturates and amphetamines at absence of false positive results in case of analysis of samples containing non-narcotic medications. The results of study of samples of dried urine spot on paper (n=50) well coincided with the results of analysis of fluid samples for all analyzed analytes. On the basis of proposed multiplex analysis a test-system Narc-Phosphan was developed for quantitative studying simultaneously up to 96 samples of various biological fluids, including as dried spots on paper. The analysis demonstrated high sensitivity, specificity and exactness during detection of the most prevailed narcotic substances that permits to propose this technique as a primary test during mass check-ups of population with purpose of detection of drug abuse, especially at the earlier stage.

Multiplex immunoassay for detection of immunoglobulin G to herpes simplex virus types 1, 2 and cytomegalovirus based on PHOSPHAN technology.

We have developed a multiplex immunoassay test (immunochip) based on PHOSPHAN technology for the detection of immunoglobulin G to herpes simplex virus (HSV) types 1, 2 and cytomegalovirus (CMV). The immunochip consists of HSV type specific gG1 (HSV-1) and gG2 (HSV-2) recombinant antigens, the lysate antigen for detection of total IgG to both HSV types (HSV 1/2), and CMV specific chimeric recombinant antigen containing the immunodominant sequences of pp150, gB, pp28 and pp52 proteins. The sensitivity and specificity of simultaneous IgGs detection with recombinant proteins were comparable to the commercial ELISA kits regardless of the kind of investigated serum specimens (patient sera, standard serum panels). The lysate HSV antigen was as sensitive but significantly less specific, so that it could not be recommended for use as a component of the multiplex test. These results can be used as a basis for creating commercial multiplex tests intended for high-productive screening of HSV, CMV and other TORCH-infections in a clinical laboratory.

[The detection of immunoglobulins G to agents of TORCH-infections using technique of multiplex immunoassay on the basis of FOSFANtm technology.]

The immune chip is developed for detecting immunoglobulins G to agents of four infections of TORCH-complex (toxoplasmosis, German measles, cytomegalovirus and herpes viral infections) on the basis of FOSFANtm technology. The sensitivity and specificity of simultaneous detection of IgG on immune chip were comparable with indices of commercial immunoenzyme test-systems, including under analysis of standard panels of serums. This permits considering derived results as a basis for development of commercial multiplex test intended for highly productive screening of TORCH-infections in clinical laboratory.

[THE APPLICATION OF TECHNOLOGY OF IMMUNE CHIPS FOSFANTM FOR INVESTIGATION OF MARKERS OF THYROID GLAND].

The set of reagents was developed on the basis of technology of immune chips in flatbed format FOSFANTM to apply in multiplex detection of markers of functional state of thyroid gland. It is demonstrated that the set permits carrying out quantitative measurements of level of free thyroxine and thyrotropic hormone in human blood serum within clinically valuable ranges of concentration. The high sensitivity and good concordance with results of immune enzyme analysis under examination of clinical samples were demonstrated.

[THE SELECTION OF ANTIGENS FOR DETECTING OF IMMUNOGLOBULIN G TO CYTOMEGALOVIRUS ON THE BASIS OF FOSFAN TECHNOLOGY].

The results of selection of composition of antigens to cytomegalovirus in the structure of multiplex test on the basis of FOSFAN™ technique are presented. In the process of detection of immunoglobulin G (IgG) to this virus the best indicators of sensitivity were registered with mosaic antigen containing immunodominant sequences of proteins pp150, gB, pp28 and pp52; reliably lower indicators of sensitivity was registered with phosphoprotein pp150; the lowest indicators of sensitivity were registered with proteins gB and pp65. The specificity made up from 93.5% to 96.8% independently of type of antigen. The mosaic antigen ensured the best ratio between sensitivity and specificity of immunoassay and is considered as the main component of immunochip for detecting IgG to cytomegalovirus.

[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.

[Lanthanide fluorescence immunoassay of 17alpha-hydroxyprogesterone in dried blood samples for neonatal screening for adrenogenital syndrome].

The first Russian assay of 17alpha-hydroxyprogesterone in dried blood spots has been developed to use for neonatal screening for adrenogenital syndrome (AGS). The technique is modeled on solid-phase lanthanide fluorescence immunoassay with time-resolution detection and it ensures the hormone to be determined in a 3.2-mm dried blood spot in the concentration range of 0 to 400 nmol/l, the coefficient of variation being not greater than 15%, and the results correlated with those of the DELFIA Neo170HP test system. The tests of 387 dried blood samples carried out in three regions have demonstrated the efficiency of the technique for screening and verifying neonatal AGS.

[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.

[Prospects of the integration of dry blood spot technology with human health and environmental population studies].

This literature review is dedicated to prospects of the use of whole blood dried on special filter paper as a source of biological material for human health and environmental population studies. Evident advantages of this low-invasive approach include the following: it is easy to take a blood sample from a patient's finger ofa neonate's heel; the cost of sampling as well as transportation and storage of samples is low; paper blanks are safe to manipulate with and convenient to mail in sealed plastic packages. Many analytes, such as DNA, become more stable after drying, which allows for the detection of phenotypic and genotypic markers, as well as multiple gene mutations by multiplex DNA amplification. Modern diagnostic techniques make it possible to detect a wide spectrum of biomarkers characterizing the condition of the endocrine, cardiovascular, reproductive, and immune systems of the organism in a single drop of blood. This allows considering paper blanks with dry blood the key component of multilevel interdisciplinary population studies on neonatal screening, disease spread surveillance, seroepidemiological monitoring, and ecological and genetic research.

[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.

[The lanthanide fluorescence immunoassay of the total thyroxin in dried bloodstains on paper]. / Lantanidnyi immunofliuorestsentnyi analiz obshchego tiroksina v vysushennykh na bumage piatnakh krovi.

The lanthanide fluorescence immunoassay was elaborated for quantitative determination of the total thyroxin T4 in bloodstain dried in filter paper; the fields of its clinical application were defined. The method is based on the hardphase concurrent immunoassay with specific monoclonal antibodies to T4 marked by chelates of europium ions and with conjugate of the T3 heterologous hapten sorbed in plate holes with bovine serum albumin. Measurements of the fluorescence intensity were made by a fluorometer in the time resolution mode. The method ensures the T4 determination in a dry bloodstain with a diameter of 3.4 mm within the concentration range of 0 to 400 nmol/l and with the variation coefficient of below or equal to 15%; the results correlated with the findings of the T4 analysis by the DELFIA Neo T4 set, "Wallac Oy", Finland. The method efficiency was demonstrated for screening and verifying the congenital thyroid deficiency in newborns; it was also confirmed that the method can be used for monitoring the functional thyroid condition in adult patients.

[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.

Epitope mapping of the outer surface protein A (OspA) of the spirochete Borrelia burgdorferi using a panel of monoclonal antibodies and lanthanide competition fluoroimmunoassay.

A panel of fourteen different monoclonal antibodies was used for detection and analysis of antigenic determinants located on the outer surface protein A (OspA) of the spirochete Borrelia burgdorferi, which is a causative agent of tick-borne borreliosis (Lyme disease). Two main and several minor partially overlapping antigenic determinants have been found on the surface of the OspA protein of Borrelia burgdorferi sensu stricto (strain 297) by lanthanide competition fluoroimmunoassay. One of the main antigenic determinants is located in the N- and the other in the C-half of the OspA molecule. The involvement of the OspA protein in intact Borrelia burgdorferi sensu stricto (four bacterial strains have been analyzed: 297, B31, FR90-594, and CA90-742) is associated with retention of the above-mentioned two major antigenic determinants, but unlike the case of the isolated OspA they are partially overlapping with each other and with other antigenic determinants. The protein of the spirochete Borrelia afzelii (two bacterial strains have been analyzed: Ip-21 and Pko) contains only one antigenic determinant, which is the same as the main determinant of the OspA protein of Borrelia burgdorferi sensu stricto located in the N-half of the OspA molecule.

[The outlook for an improvement in the agents for detection based on immunofluorescence analysis]. / Perspektivy sovershenstvovaniia sredstv indikatsii na osnove immunofliuorestsentnogo analiza.

The review deals with prospects for improving the agents for rapid indication of biological agents by a combination of currently available immunochemical and molecular genetic analyses and the latest developments in the design of luminescent tracers, techniques of recording superweak luminous fluxes in the photon count mode and laser spectroscopies. Immunosensor technologies and homogeneous immunoassay are shown to be optimal for the studies to rapidly detect pathogens and toxins. It is most advisable to employ highly sensitive, specific, and efficient methods of molecular hybridization and multicomponent solid-phase immunoassay by recording biospecific binding products in the mode of time luminescence resolution for environmental studies and clinical laboratory diagnoses in resident laboratories. Continuous water and air follow-up monitoring involves the analysis by using immunosorbents, as well as immunosensors and flow cell fluorimetry is noted to become an important independent line in the development of biological indication agents under the increasing man-made influence of the environment.

[Lanthanide immunofluorescent analysis: virus detection and the serodiagnosis of viral infections]. / Lantanidnyi immunofliuorestsentnyi analiz: indikatsiia virusov, serodiagnostika virusnykh infektsii.

This communication summarizes 10-year experience gained by the author in developing and using the lanthanide immunofluorescence assay (LIFA) for the laboratory diagnosis of viral infections. The bulk of studies has been conducted on natural focal viruses, including Venezuelan equine encephalitis, tick-borne encephalitis, Crimean Congo hemorrhagic fever, California serogroup, and other viruses. Moreover, test systems have been developed for diagnosis of infections caused by herpes simplex and cytomegaloviruses. The studies performed have demonstrated the sensitivity of LIFA in the indication of viruses in the laboratory materials and the samples from natural foci is 10-100 times higher than that of enzyme immunoassay and it is close to that of the biological isolation assay; the specificity of LIFA is comparable to that of the neutralization reaction, but it is more accessible in practice due to the fact it does not require the use of living viruses and biological models. The results of detection of herpes viruses in the clinical samples by LIFA are shown to rather well correlate with the data of virus isolation in the cultured cells, with other diagnostic methods and with the clinical manifestations of diseases. LIFA is recommended for use in large-scale studies involving the monitoring of infection foci and the screening of risk population groups for social infectious diseases.

[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.