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A Phosphorescence Analysis (PHOSPHAN)-based test system was developed for simultaneous detection of thyroid-stimulating hormone (TSH) and free thyroxin (fT4), the markers of thyroid function, in dried human blood samples. The immunoassay was performed at the bottom of polystyrene 96-well microplate wells covered with analyte-specific microarrays (0.35 mm in diameter). The detection system consisted of polystyrene nanoparticles (46 ± 14 nm in diameter) with incorporated long luminescent Eu(III) chelate complexes (1960 molecules/particle). Fluorescence intensity was measured in time-resolved mode (TRF). Analytical parameter values and possible limitations of this experimental system were investigated. The system allowed the simultaneous detection of high-molecular-weight and low-molecular-weight compounds in clinically relevant concentration ranges, from 2.0 to 50 pmol/L for free T4, and 0.8 to 50 µIU for TSH. The sensitivity of the analysis was 1.0 ± 0.2 pmol/L (fT4) and 0.3 ± 0.1 µIU/mL (TSH), which corresponded to the detection thresholds of 3 × 10–18 mol T4 and 0.9 × 10–9 IU TSH. The test determined correctly the TSH and T4 concentrations in standard samples used in quality assessment programs, as well as the thyroid status of the patients included in the investigation (n = 48). Results of hormone detection in dried blood spots with our experimental test system correlated well with the results of hormone detection in serum samples using commercial kits (r = 0.9730 for TSH and r = 0.8585 for fT4). The main drawback of the system was associated with high variability of measurement results (up to 20%), which limits the possibility of its application for the quantification of the hormones level in patient blood. We believe that this system can be useful for detecting thyroid disorders both in the existing programs of neonatal screening for congenital hypothyroidism and in mass screening surveys in risk groups of the adult population, for example, in ecologically unfavorable regions.

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.

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.

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.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.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.

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).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.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.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.

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.

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 (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.

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.

Two variants (direct and indirect) of enzyme linked immunosorbent assay (ELISA) of phenobarbital are compared. Both techniques were developed on the basis of the same monoclonal antibodies, and horse radish peroxidase was used as the label in both cases. When microtitration plates are used as the solid phase, indirect ELISA, in which phenobarbital of the sample competes with phenobarbital sorbed on plates in the form of a conjugate with protein for the binding with peroxidase-labeled antiphenobarbital antibodies, is preferable. In indirect ELISA, the sample volume was 5 microliters, the time of assay was 40 min, the variability coefficient was8%.

Monoclonal antibodies to phenobarbital have been developed. Spleen cells of BALB/c mouse immunized with conjugate keyhole limpet haemocyanin and phenobarbital were fused with P3-X63-Ag8.653 mouse myeloma cells. Three monoclonal antibodies, selected by indirect ELISA, were produced in mouse ascite fluids, purified and analyzed. Antibody 1A9 was selected for use in immunoassay, its association constant being 1.6 x 10(9) M-1.