Homogeneous immunoenzyme analysis of human immunoglobulin G using horseradish peroxidase

The author studied the steady-state kinetics of cooxidation of 4-aminoantipyrine (AAP) with phenol and its derivatives--alpha-naphthol, o- and m-acetylaminophenols--by horseradish peroxidase and its conjugate with human immunoglobulins IgG (HRP-IgG). When phenol and AAP were used as peroxidase substrates, anti-human IgG antibodies stimulated the HPR-IgG enzyme activity in the presence of excess H2O2. A homogeneous enzyme immunoassay of human IgG was developed on the basis of this stimulating effect. The kinetics of the interactions between immunological reagents was studied. In the presence of 3% polyethylene glycol 6000, a complete antibody-antigen interaction proceeds at 37 degrees for 15-20 min. The sensitivity of the enzyme immunoassay is 350 ng/ml IgG, and the dynamic detection range is 0.35-15.0 mg/ml.     

The diagnosis of antileptospiral antibodies in human subjects by solid-phase immunoenzyme analysis

The possibility of using the enzyme-linked immunosorbent assay (ELISA) for the diagnosis of leptospirosis has been shown. This method has proved to be more simple and sensitive than the leptospiral microagglutination and lysis test. The data on obtaining genus-specific leptospiral antigens are presented. As revealed in this study, the antigens obtained by the complex treatment of microbial cells with ultrasound and detergents show the maximum activity in ELISA. The optimum parameters of the ELISA system for the diagnosis of leptospirosis have been established.     

Patterns in the immunoenzyme analysis of bacterial cells

Some regularities of the enzyme immunoassay (EIA) of whole bacterial cells have been studied on one of the bacillary species of contaminant microflora. Early detection of this microorganism is highly important for the microbiological production of alpha-amylase and alkaline protease (produced by Bacillus subtilis). The effective kinetic and equilibrant parameters of the interaction of peroxidase-labeled antibodies with the cells of the contaminant microflora in the solution and on the surface of the polystyrene plates have been defined. Two methods for the separation of cells after their interaction with peroxidase-labeled antibodies have been optimized: filtration involving the use of special filter plates and centrifugation in plates. The method for the immobilization of cells in the wells of standard assay plates by centrifugation has been proposed. Four EIA methods for measurement of contaminant microflora have been developed and optimized. These methods permit the determination of the microflora at concentration of 5 X 10(5)-5 X 10(4) cells/ml, depending on the scheme of the assay, within 1-3.5 minutes.     

The immunoenzyme analysis of ceruloplasmin

A highly specific and sensitive enzyme immunoassay (EIA) system, suitable for the qualitative analysis of ceruloplasmin, has been developed. The possibility of its use for the examination of children with mononucleosis and pseudotuberculosis has been studied. An increase in the concentration of ceruloplasmin has been more pronounced in infectious mononucleosis (0.506 +/- 0.026 g/l) and pseudotuberculosis (0.421 +/- 0.157 g/l). The results of EIA coincided with the data obtained by radial immunodiffusion.     

Dot immunoenzyme analysis in biological research

The modern state, general methodologic problems and the possibilities to use in biological research the dot enzyme-linked immunosorbent assay (dot-ELISA) are analysed in the review. New types of microporous polymer membranes and equipment for the application of the solid-phase reagent and performing the assay are considered. Different variants of dot-ELISA and methods for the evaluation of results obtained in the assay as well as the ways for its optimization are discussed.     

Use of immunoenzyme analysis in the diagnosis of toxoplasmosis

The method for the preparation of antigen for ELISA and the technique of this assay are described. The comparative data obtained in the examination of 50 donors and 95 children suspected to have toxoplasmosis, carried out with the use of immunofluorescence and ELISA, are presented. A good correlation between the results of the both tests has been shown.     

Development of a Soviet immunoenzyme test system for the quantitative determination of human immunoglobulin E

An EIA system for the quantitative determination of human IgE was developed. As the solid phase, polystyrene microplates sensitized with the gamma globulin fraction of sheep antiserum to human IgE were used in this system. Peroxidase conjugate with IgE was prepared with the use of periodate technique. The optimum parameters for the sorption of antibodies on polystyrene were determined: protein concentration, the pH of the buffer, temperature, the time of fixation, the working dilution of the conjugate. As the substrate of peroxidase activity, ortho-phenilenediamine solution with hydrogen peroxide was used. The result was evaluated by means of a photometer, model Titertek Multiskan MC (Flow Laboratories Ltd., Great Britain), at the wavelength 429 nm. The new EIA system proved to be specific and detected IgE in the sera under investigation at a concentration of 10(-9) g/ml. The system permitted the determination of the normal level of IgE in a group of healthy adults, this level being, on the average, 76 +/- 9 kU/1.     

Use of cooperative monoclonal antibodies in immunoenzyme analysis for determining human tumor necrosis factor

A cooperative sandwich enzyme immunoassay (EIA) based on the newly produced pair of cooperative monoclonal antibodies (mAbs) against human tumor necrosis factor (TNF) was developed and characterized. It was found that, when used simultaneously, cooperative mAbs was capable to bind TNF from its preformed complexes with soluble TNF receptors (sTNF-R), thus providing the effective TNF detection in ex vivo samples by the respective one-step cooperative EIA. While demonstrating typical analytical characteristics regarding variability, dynamic range and specificity, a cooperative EIA offers an advantageous combination of high sensitivity (< 2 pg/ml) and short-time TNF capture protocol (1 hour). Application of cooperative EIA for TNF detection in clinical samples has demonstrated an increased serum TNF levels in patients with the mixed connective disease and infectious endocarditis that positively correlated with severity of systemic inflammatory reactions. Production and EIA application of cooperative mAbs would be promising in development of standardized and clinically applicable immunoassays for cytokines.     

Rapid serological screening of human sera for the presence of measles virus antibodies in solid-phase immunoenzyme analysis

A single dilution technique has been used for the determination of antimeasles antibody titer. The method involved the plotting of the calibration curve and the characterization of the serum by arbitrary "evaluation units" in comparison with the specially selected positive serum whose titer was taken to be equal to 100 "evaluation units". By means of this method 57 sera obtained from children immunized against measles and 118 sera from non-vaccinated adults aged 18-22 years were examined. The values of the calculated titers were similar to those determined experimentally. This recommends this method for seroepidemiological investigations aimed at determining the level of herd immunity to measles.     

Solid-phase immunoenzyme analysis of Coccidioides immitis antigens

A highly effective and specific solid-phase enzyme immunoassay system has been developed. The enzyme immunoassay is a highly sensitive technique for the detection and identification of C. immitis cellular and metabolic antigens. This technique is suitable for the study of strain differences in the antigenic composition of C. immitis, rendered harmless by different methods. The expediency of the preliminary sonification of cell suspensions of C. immitis, the causative agent of coccidioidomycosis, has been experimentally confirmed.     

Magnetic immunoenzyme analysis of Yersinia pestis antigens

The detection of Y. pestis cells in magnetic enzyme immunoassay is carried out with the use of magnetic polyacrylamide microgranules. In the assay system for the determination of the antigen commercial Y. pestis antigens, peroxidase-labeled antibodies, the substrate mixture consisting of sodium salt of 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid and H2O2 in citrate-phosphate buffer solution, pH 4.5, are used. The sensitivity of the method is 5 X 10(4) microbial bodies per ml.     

Rapid immunoenzyme diagnosis of influenza in patients with various allergoses and bronchial asthma

In an investigation carried out in the allergological clinic of the Tbilisi State Medical Institute in the period between two outbreaks of influenza, the presence of influenza antigen was determined in nasal washings taken from 127 patients with different allergoses and bronchial asthma by means of the enzyme immuno-assay with the use of the type-specific virion antigen of M1-protein. This method was found to be highly sensitive and to have some advantages over traditional methods used for the diagnosis of influenza. In patients with preasthma and different forms of bronchial asthma elevated susceptibility to influenza infection and its unfavorable influence on the clinical course of these pathological conditions were established.     

Sorption capacity of polystyrene plates used in immunoenzyme analysis

The data indicating that the degree of sorption of the antigen onto the plate depends on the duration of this process are presented. As shown in this investigation, the lower the adsorption activity of the plates, the longer the process of the sensitization of plates with the antigen. The process of the sorption of the antigen onto the plates with low adsorption capacity may be accelerated by sensitizing the plates at different temperatures. Besides, the adsorption capacity of the plates was increased by ultraviolet irradiation.     

Phenological modifications in plants by various edaphic factors

Various mechanical, chemical and physical soil analyses were carried out, in addition to weather observations, for 3 years at several sites along an oceanic-continental gradient in a fjord district in western Norway. All the environmental factors observed were correlated with the spring and a few late-season phenophases of many native and cultivated woody plants and some herbs by simple, linear correlations and by stepwise multiple and partial analyses. Different techniques were used to try and eliminate many intercorrelations between various environmental factors. As expected, air temperature measurements in nearly all analyses from these temperate region districts gave the most significant correlations with the phenology of the plants, the temperature during the night generally being the most important in mainly vegetative periods, e.g. to leaf bud break in spring, and the temperature during the day affecting the more generative phases, such as the period between leaf bud break and flowering. The other environmental factors, however, showed strong variation in correlation significance among the various species studied and also with different phenophases of the same species. Various hypotheses are put forward to explain such variation. Air humidity (including precipitation) and /or soil moisture (including intercorrelated parameters, e.g. soil grain size and bulk density) were relatively often found to be of importance. In the stepwise multiple analyses for leaf bud break of the birch (Betula pubescens), for instance, the amount of precipitation was the second factor to enter the analyses by a positive correlation with the developmental rate, after the most important factor, the night temperature. Positive correlations with a high clay content and bulk density in the soil indicated that high soil moisture is also favourable for early bud break in the birch. Other phenophases that seemed to be favoured by a good water supply were leaf bud break of the bird cherry (Prunus padus) and rowan (Sorbus aucuparia), and flowering of the hazel (Corylus avellana), common lilac (Syringa vulgaris), plum (’Victoria’) and currant (’Red Dutch’) and also, to some degree, the goat willow (Salix caprea). The amount of ions (P, K, Mg and Ca) often showed negative correlations with the developmental rate, particularly of earlier phenophases of both native and cultivated plants (except for the apple ’Gravenstein’ and pear ’Moltke’), possibly, indicating that a high nutrient level delayed plant development. A similar explanation might be given for the observation that high pH in the soil often seemed to delay plant development (leaf bud break of Betula, Sorbus, Syringa and plum, and flowering of Corylus, bluebell (Campanula rotundifolia) and red currant). According to the analyses there seemed to be a tendency for plants that are particularly dependent on warm weather for leaf bud break, e.g. the ash (Fraxinus excelsior), and flowering, e.g. Prunus, pear, apple and, to some degree, the raspberry (’Preussen’), to be less dependent on other environmental factors for their development. For instance, if there were any effects of water for these plants, they were negative for moisture and soil factors intercorrelated with water. Received: 25 October 2000 / Revised: 9 May 2001 / Accepted: 24 May 2001     

An immunoenzyme test system for determining gaprin in the air]

A highly specific and sensitive enzyme immunoassay system for the quantitative determination of haprin in the air at a concentration of not less than 0.0001 mg of haprin per m3 of air has been developed. The assay system has been approved at the Svetloiarsk plant for the production of vitamin protein concentrate and can be used for the control and evaluation of haprin pollution of the environment at areas adjoining microbiological plants.     

Use of solid-phase immunoenzyme analysis for the serodiagnosis of pseudotuberculosis

The diagnostic test system based on the solid-phase enzyme immunoassay (EIA) for the detection of antibodies to Yersinia pseudotuberculosis in the sera of patients with the use of Soviet-made preparations and reagents has been developed. The test has been performed in microchambers for immunological reactions, thus making it possible to decrease the consumption of reagents 10-20 times in comparison with the traditional technique with the use of plates. The results of the titration of 42 sera in EIA and in the passive hemagglutination test (PHAT) are indicative of the presence of positive correlation (r = 0.78; p less than 0.05) between antibody titers in EIA and PHAT. A fourfold or greater increase in antibody titers has been determined by means of EIA in 80% of cases and with the use of PHAT in 55% of cases. The minimum diagnostic titer yielded by EIA has been determined: 1:256.     

The use of immunoenzyme analysis in the laboratory diagnosis of plague]

The possibility of using the enzyme immunoassay (EIA) for the early diagnosis of pneumonic plague was studied in experiments on monkeys. EIA was shown to be more effective than the passive hemagglutination test. The diagnostic value of blood serum samples was found to be higher than that of nasopharyngeal mucus samples taken from the sick animals. The conclusion on the suitability of EIA for the early laboratory diagnosis of this disease was made.