[Immunoassay of nine serological tumor markers on hydrogel-based microchip].

A prototype of test-system for simultaneous quantitative assay of nine tumor markers in blood serum was developed. The main constituent of the test-system is OM-9 biochip containing immobilized antibodies against nine oncomarkers: α-fetoprotein (AFP), carcinoembryonic antigen (CEA), human chorionic gonadotropin (HCG), cancer antigen 15-3 (CA 15-3), cancer antigen 125 (CA 125), cancer antigen 19-9 (CA 19-9), prostate-specific antigen, total (PSAtot) and free (PSAfree) forms, neuron-specific enolase (NSE). The biochip-based assay procedure for carrying out simultaneous quantitative determination of nine tumor markers in patient's blood serum: two-steps sandwich-immunoassay, was proposed. The main analytical characteristics of the method were obtained. The results permit to consider the prototype of the test-system as a promising instrument for clinical application. The test-system prototype was tested using blood serum samples of oncological patients (252 samples) and healthy donors (185 samples). Increased concentrations of one or more tumor markers above the normal level were found in 76.6% cases of oncological patients and only in 6% cases of healthy donors. For colorectal cancer patients group, application of modern statistical methods of data-processing in medical researchers, i.e. ROC-analysis and logistic regression, indicted that the simultaneous assay of nine tumor markers on biochips showed much more diagnostic significance (area under the ROC-curve (AUC) reached 0.84) than traditional assay of 2 tumor markers, CEA and CA 19-9 (AUC = 0.59). The developed biochip-based test-system can be recommended both for the estimation of people's health, e.g., for standard medical examination, and for tracking of tumoral process in postsurgical period or after specific tumor treatment.

[Sequence-specificity of protein-oligonucleotide interactions and development for protein isolation using sorptive medium with immobilized protein-specific oligonucleotides].

Sequence-specificity of binding of ribonuclease binase to oligodeoxyribonucleotides immobilized in biochip gel pads was studied. Binding constants for the complexes between binase and selected oligonucleotides were measured. Oligodeoxyribonucleotides GAGAGAG and GAGAGAGAG were found to be high-specific in interaction with binase. These oligonucleotides were used as molecular probes for immobilization in a sorptive medium for subsequent isolation and concentrating of binase from diluted water solutions. Volume capacity of the developed sorptive mediums containing immobilized oligodeoxyribonucleotides GAGAGAG and GAGAGAGAG was found to be 2.6 and 2.3 mg of binase per 1 mL of sorbate correspondingly. After the procedure of affinity chromatography and elution ofbinase from sorptive medium the protein showed the same affinity of binding to oligonucleotides as the initial sample.

[Preparation and characterization of monoclonal antibodies to the cholera toxin].

Monoclonal antibodies to cholera toxin were obtained. They do not cross-react with the termolabile toxin (LT) of Escherichia coli, ricin, diphtherial toxin, staphylococcus enterotoxins of SEA, SEB, SEI, SEG, or the lethal factor and protective antigen of the anthrax toxin. Pairs of antibodies for the quantitative measurement of the cholera toxin in sandwich enzyme immunoassay (EIA) were selected. The detection limit of the toxin is 0.2 ng/ml for plate EIA and 0.44 ng/ml for microchip EIA. The presence of milk, broth, or surface water in the toxin samples does not reduce the sensitivity of EIA.

[Production and characteristics of monoclonal antibodies to the diphtheria toxin].

Monoclonal antibodies to the diphtheria toxin were produced without cross reactivity with the thermolabile toxin (LT) from Escherichia coli; ricin; choleraic toxin; the SeA, SeB, SeE, SeI, and SeG toxins of staphylococcus; the lethal factor of the anthrax toxin; and the protective antigen of the anthrax toxin. A pair of antibodies for the quantitative determination of the diphtheria toxin in the sandwich variation of enzyme-linked immunosorbent assay (ELISA) was chosen. The determination limit of the toxin was 0.7 ng/ml in plate and 1.6 ng/ml in microchip ELISA. The presence of a secretion from the nasopharynx lavage did not decrease the sensitivity of the toxin determination by sandwich ELISA. The immunization of mice with the diphtheria toxin and with a conjugate of the diphtheria toxin with polystyrene microspheres demonstrated that the conjugate immunization resulted in the formation of hybridoma clones which produced antibodies only to the epitopes of the A fragment of the diphtheria toxin. The immunization with the native toxin caused the production of hybridoma clones which predominantly produced antibodies to the epitopes of the B fragment.

[Development of a biochip for quantitative determination of two forms of prostate specific antigen using internal calibration curve].

Three-dimensional gel-based microchip allowing simultaneous quantitative detection of total (PSAtot) and free (PSAfree) forms of prostate specific antigen in human serum (in a format "one patient-one biochip") was developed. A method, which doesn't require preliminary construction of calibration curves when performing an assay, was applied for quantitative determination of PSAtot and PSAfree. Gel elements with immobilized antigen (PSA) in different concentration, forming an internal calibration curve, were included in a structure of the microchip, in addition to the elements with immobilized antibodies specific against PSAtot and PSAfree. The specialized software "ImaGelAssay" was used for data processing and interpretation. The sensitivity of the assay performed on biochips was 0.3 ng/ml for PSAtot and 0.2 ng/ml for PSAfree. Variation coefficient for the measurements inside one series of microchips didn't exceed 10%. Correlation coefficient between the results of measurements in human sera obtained on biochips and by the standard ELISA method was 0.988 for PSAtot and 0.987 for PSAfree.

[Dye with low specificity to nucleotide sequences of DNA: use for assessing the quantity of oligonucleotides, immobilized in cells of biological microchips]. / Kasitel' s nizkoi spetsifichnost'iu k nukleotidnoi posledovatel'nosti DNK: primenenie dlia ostenki kolichestva oligonukleotidov, immobilizovannykh v iacheikakh biologicheskikh mikrochipov.

To assess the DNA amount in samples (e.g., in biological microchip gel pads) by means of fluorescent dyes, one should use the dyes whose fluorescence weakly depends on DNA composition and structure. With the ImD-310 dye created for this purpose, we have analyzed the staining of single- and double-stranded oligo- and polynucleotides of different nucleotide composition, length, and concentration both in solution and being immobilized in biological microchip gel pads. It turned out that ImD-310 has no pronounced specificity to the single- and double-stranded nucleotide sequences, while the intensity of fluorescence for the dye complexes with d(A)8, d(T)8, d(C)8, and d(G)8 at high temperatures (50 degrees C) differs by less than 25%. A linear correlation has been established between the intensity of fluorescence and the amount of oligonucleotides immobilized on a biological microchip. The plots of the intensity of fluorescence against the concentration of NaCl and the temperature were obtained. By using a generic microchip containing all 4096 hexamer oligonucleotides, it has been determined that the dye has no distinct specificity to any certain motifs of the nucleotide sequence. Thus, ImD-310 may serve as an efficient fluorescent probe to quickly estimate the amount of oligonucleotides immobilized in a microchip, in an electrophoretic gel, etc.

[Microchips based on three dimensional gel cells: history and perspective]. / Mikrochipy na osnove trekhmernykh iacheek gelia: istoriia i perspektivy.

The review describes the history of creation and development of the microchip technology and its role in the human genome project in Russia. The emphasis is placed on the three-dimensional gel-based microchips developed at the Center of Biological Microchips headed by A.D. Mirzabekov since 1988. The gel-based chips of the last generation, IMAGE chips (Immobilized Micro Array of Gel Elements), have a number of advantages over the previous versions. The microchips are manufactured by photo-initiated copolymerization of gel components and immobilized molecules (DNA, proteins, and ligands). This ensures an even distribution of the immobilized probe throughout the microchip gel element with a high yield (about 50% for oligonucleotides). The use of methacrylamide as a main component of the polymerization mixture resulted in a substantial increase of gel porosity without affecting its mechanical strength and stability, which allowed one to work with the DNA fragments of up to 500 nt in length, as well as with rather large protein molecules. At present, the gel-based microchips are widely applied to address different problems. The generic microchips containing a complete set of possible hexanucleotides are used to reveal the DNA motifs binding with different proteins and to study the DNA-protein interactions. The oligonucleotide microchips are a cheap and reliable tool of diagnostics designed for mass application. Biochips have been developed for identification of the tuberculosis pathogen and its antibiotic-resistant forms; for diagnostics of orthopoxviruses, including the smallpox virus; for diagnostics of the anthrax pathogen; and for identification of chromosomal rearrangements in leukemia patients. The protein microchips can be adapted for further use in proteomics. Bacterial and yeast cells were also immobilized in the gel, maintaining their viability, which open a wide potential for creation biosensors on the basis of microchips.

[Solid-phase synthesis of peptides containing arginine with an unprotected guanidine group]. / Tverdofaznyi sintez peptidov, soderzhashchikh arginin s nezashchishchennoi guanidinovoi gruppoi.

A new variant of the solid phase synthesis of arginine-containing peptides was proposed. The conditions for the attachment to the Wang polymer of N alpha-Fmoc-arginine containing a protonated guanidine group were found. We demonstrated that this attachment is accompanied by neither racemization nor the attachment of the second Arg residue. Side reactions involving the guanidine group of arginine were studied, and methods for their prevention were proposed. The comparison of the carbodiimide method with a 1-hydroxybenzotriazole additive and a modified method with the use of Kastro's reagent for the introduction of N alpha-Fmoc-Arg residue with the unprotected guanidine group into the growing peptide chain demonstrated the advantages of the second method. Bradykinin and a peptide corresponding to the 584-591 sequence of the transmembrane gp41 from HIV-1 were synthesized by the method proposed here.

[The dynamic changes in the tissue components of the myocardial layers under compensatory heart hypertrophy in exposure to a peptide hydra morphogen]. / Dinamika izmenenii tkanevykh komponentov sloev miokarda v usloviiakh kompensatornoi gipertrofii serdtsa pri vozdeistvii peptidnogo morfogena gidry.

Morphometric analysis of the left ventricle myocardial layers under heart hypertrophy was carried out. Values of tissue parameters on the 3rd, 10th and 24th days after 50% coarctation of the abdominal aorta were received. Is shown the heart functioning under conditions of myocardial compensatory hypertrophy development is characterized by expressed zones and different directions of changes of quantitative parameters of tissue components. The single injection of HMP (Hydra morphogen peptide) made the development of myocardial hypertrophy easy at early stages, decreasing depth of manifestations morphologic changes and perhaps affected the time of their development.

[Effect of peptide hydra morphogen on the structure of tissue components of rat myocardial layers in the early period of heart hypertrophy development]. / Vliianie peptidnogo morfogena gidry na strukturu tkanevykh komponentov sloev miokarda krysy v rannie sroki razvitiia gipertrofii serdtsa.

The morphological study showed that the single injection of HMP (Hydra Morphogen Peptide) facilitated the development of the myocardium hypertrophy. This effect is expressed in the subendocardial layer most of all, where the normalization of the relative volume and diameter of the cardiomyocytes and the decrease of the connective tissue oedema are noted. These parameters are normalized in the intramural and subepicardial layers in less degree. The decrease of the connective tissue oedema is noted in the intramural layer. The normal correlation recovery between the relative volumes of the cardiomyocytes and the connective tissue is shown in the subepicardial layer, the diameters of the myocytes are essentially increased. The influence of HMP is manifested not only on the muscular and connective tissue components of the myocardium, but touches on also the system of the myocardial wall vessels, causing the redistribution of the intramural blood circulation.

[Effect of a new peptide morphogen on androgen metabolism in the gonads during sexual maturation in the rat]. / Vliianie novogo peptidnogo morfogena na metabolizm androgenov v gonadakh pri polovom sozrevanii krys.

After chronic administration of a new neuropeptide (hydra growth activator--HGA) to male and female Wistar rats from the 29th to 43rd day of life at a dose of 30 micrograms X kg-1 of body mass a study was made of [3H] testosterone, [3H] 5 alpha-dehydrotestosterone [( 3H] DHT) and [3H] 5 alpha-androstane-3 beta, 17 beta-diol metabolism in gonadal homogenates of the animals. In testis homogenates HGA administration was shown to influence the production of [3H] 3 beta-diol from [3H] DHT only. In female rats HGA administration made an effect on the majority of enzymes of androgen metabolism in the ovaries of animals with the closed and open vagina. Basing on changes in enzymatic activity of androgen metabolism in the gonads of rats in chronic HGA administration in the period of male pubescence a conclusion was made that it acted as a pubescence accelerating factor.

[Modulation of ornithine decarboxylase activity in the normal and regenerating rat liver by different doses of hydra peptide morphogen]. / Moduliatsiia aktivnosti ornitindekarboksilazy v normal'noi i regeneriruiushchei pecheni krys razlichnymi dozami peptidnogo morfogena gidry.

The authors studied the anabolic effect of peptide morphogen of the hydra undecapeptide on normal and regenerating rat liver. Ornithine decarboxylase (EC 4.1.1.17) activity served as a marker. Intraperitoneal injection of the peptide into intact animals stimulated ornithine decarboxylase activity in a dose-dependent manner. In partially hepatectomized rats the peptide stimulated ornithine decarboxylase activity in the dose of 20 micrograms/kg body weight while greater doses inhibited the enzyme activity.

[Effect of low doses of the hydra peptide morphogen on protein biosynthesis in the intact and regenerating rat liver]. / Vliianie nizkikh doz peptidnogo morfogena gidry na biosintez belka v intaktnoi i regeneriruiushchei pecheni krys.

The dependence of ornithine decarboxylase (ODC) activity on hydra peptide morphogen doses has been established. The parameters of protein synthesis were determined in normal and regenerating rat liver, using the peptide in a dose of 20 micrograms/kg body weight, initiating maximum enzyme activity. It was established that intraperitoneal injection of the peptide in partially hepatectomized animals stimulated ODS activity in dose-dependent manner and was dome-shaped. The peptide injection in intact animals does not affect the intensity of 3H-leucine inclusion into the liver protein and the protein content in rat liver. However, the peptide injection in partially hepatectomized animals increased the level of 3H-leucine inclusion into the protein of regenerating liver and stimulated protein accumulation in this type of tissue.