Sorption properties of polystyrene plates used in immunoenzyme analyses

The capacity of polystyrene carriers used in the enzyme immunoassay (EIA) for adsorbing 131I-labeled human serum albumin under different conditions has been studied, and the comparison of the plates manufactured by Dynatech AG (Switzerland) and by the Leningrad Works of Medical Polymers has been made. At the first stages of the reaction the antigen is separated from the carrier and the amount of the desorbed antigen depends on its initial dose and the dilution of the assayed sera. The irregular desorption of the antigen leads to misinterpretation of the results. Comparison of the polystyrene plates has shown that each plate is characterized by individual adsorption capacity, which impedes at present the standardization of EIA-based test systems.     

Methods of enhancing the specificity of immunoenzyme test systems for the determination of human IgE

The sensitivity and specificity of an enzyme immunoassay (EIA) system for the determination of total IgE in humans increases if polystyrene plates are sensitized with swine gamma-globulin and the conjugate is prepared with the use of sheep gamma-globulin obtained from the commercial preparation of sheep antiserum to human IgE. Such system becomes even more specific and sensitive if sheep antibodies to human IgE, purified by affinity chromatography, are used both for the sensitization of polystyrene plates and for the preparation of the conjugate. This conjugate is necessary for the development of EIA systems intended for the determination of specific human IgE-antibodies to various allergens.     

Possible diagnosis of Pseudomonas aeruginosa infection by an immunoenzyme method using pyoimmunogen as the antigen

The possibility of detecting P. aeruginosa antibodies in patients by means of indirect solid-phase EIA techniques is shown. This assay is carried out with the use of reagents produced in the USSR: polystyrene assay plates manufactured by the Lenigrad Medpolymer Works are used as carriers, P. aeruginosa vaccine (pyoimmunogen) obtained under semi-industrial conditions at the Mechnikov Central Research Institute for Vaccines and Sera is used as antigenic complex and the commercial preparation produced by the Gamaleia Research Institute of Epidemiology and Microbiology serves as conjugate. The studies have revealed that in 95% of cases the level of antibodies in the sera of patients with acute destructive pneumonia accompanied by pleural empyema, abscesses of internal organs and acute hematogenic osteomyelitis is essentially higher than the level of "normal" antibodies in healthy donors from whom biologically confirmed P. aeruginosa cultures can be isolated. In the groups of patients with similar nosological forms of diseases caused by other infective agents such difference in antibody titers is not detected. These results suggest that the detection of antibodies to P. aeruginosa in patients' sera by means of EIA can be used as an additional test for the diagnosis of P. aeruginosa infections.     

Immunoenzyme analysis using paper disks

Filter paper discs have been used in the enzyme immunoassay (EIA) as solid phase instead of polystyrene plates. The use of paper discs has made it possible to achieve a multiple increase in the sensitivity of sandwich EIA, thus permitting the detection of Yersinia pestis capsular antigen at a concentration of 0.4 ng/ml. Paper discs can be used not only for the sorption of antigen and antibodies, but also for the affinity purification of preparations.     

Detection of Legionella pneumophila antibodies in the blood serum of pneumonia patients by an immunoenzyme method

The conditions of making the enzyme immunoassay (EIA) for the detection of antibodies to L. pneumophila have been optimized. The use of L. pneumophila purified serotypic antigen at a concentration of 0.25 micrograms/ml for the sensitization of polystyrene plates has been shown to increase the sensitivity and specificity of the assay. 220 patients with severe pneumonia have been examined. As revealed in this investigation, antibodies to L. pneumophila can be detected in 12.2% of cases. A high degree of correlation (94.4%) between the results of EIA and the indirect immunofluorescence test has been shown.     

The characteristics of detecting the tick-borne encephalitis virus antigen in the ELISA and indirect hemagglutination reaction by means of scanning electron microscopy

The surface of polystyrene plates was studied at different stages of the enzyme immunoassay (EIA) and the passive hemagglutination (PHA) test by the method of scanning electron microscopy in the detection of tick-borne encephalitis (TBE) virus antigen. The study revealed that in the process of EIA larger antigens were washed away from the plate surface. The objects detected on the polystyrene surface were identified as conglomerations of the virions of TBE virus, but whole virions were shown to play no decisive role in EIA. The conclusion was made that, due to some specific features of this method, EIA was more sensitive in reaction with small antigens (individual glycoproteids, their small complexes). And, respectively, the PHA test was more sensitive in reaction with large antigenic complexes (whole virions, their conglomerations, immune complexes).     

Production of monoclonal antibodies to estriol and their application in the development of a sensitive nonisotopic immunoassay

The development of highly specific monoclonal antibodies to estriol and a nonisotopic immunoassay (EIA) for unconjugated estriol based on the use of these monoclonal antibodies have been described. The monoclonal antibodies show little cross reactivity with other steroids and steroid conjugates and can be used directly in immunoassays without any purification. The EIA described here can be performed in 96-well microtiter plates or polystyrene tubes that have been coated with estriol-bovine serum albumin conjugate. In this assay, estriol in the standard or clinical samples (serum or saliva) competes with the immobilized steroid on the plate or the tube for binding with the antibody. The assay shows good agreement with radioimmunoassay (RIA) and is highly sensitive and reliable. Since no prior processing or extraction of the clinical samples is necessary, the method is potentially applicable for routine use in fetal monitoring as well as in a steroid laboratory.     

A Stable Lyophilized Reagent for Use in a Potential Reference Assay for Quantitation of Anti-D in Immunoglobulin Products

Quality control of anti-D immunoglobulins intended for in vivo clinical use requires in vitro assay of potency. A lyophilized biotinylated monoclonal anti-D (biotinylated Brad-5; 99/698) has been evaluated for its suitability to serve as a working reagent in a competitive enzyme-linked immunoassay (EIA) for anti-D quantitation. The reagent demonstrated acceptable stability in accelerated degradation tests and following reconstitution. Twelve international laboratories obtained comparable potencies for each of nine anti-D samples using 99/698 in a standardized assay procedure using erythrocytes fixed to microtitre plates. We also describe the use of trehalose for stabilization of dried erythrocyte-coated microtitre plates.     

Direct immunoassay for detection of salmonellae in foods and feeds

A direct enzyme immunoassay (EIA) with polyclonal antibodies was developed for detecting salmonellae in foods and feeds. Salmonella cells were attached firmly to the wells of polystyrene microtitration plates with a capture-antibody technique. Spicer-Edwards anti-H immunoglobulin G was bound to protein A-beta-D-galactosidase to serve as the signal; 4-methylumbelliferyl-beta-D-galactoside was used as the substrate. The sensitivity threshold was 10(7) cells per ml. Direct EIA, indirect EIA, and pure-culture techniques were compared by using 48 samples of naturally contaminated foods and feeds. The direct EIA was more sensitive than the indirect EIA or pure-culture technique. Food samples were analyzed within 3 working days, and 32 samples were tested simultaneously in a single 96-well microtitration plate. False-positive or false-negative results did not pose a problem. This direct EIA is sensitive, rapid, and amenable to automation.     

Direct immunoassay for detection of salmonellae in foods and feeds.

A direct enzyme immunoassay (EIA) with polyclonal antibodies was developed for detecting salmonellae in foods and feeds. Salmonella cells were attached firmly to the wells of polystyrene microtitration plates with a capture-antibody technique. Spicer-Edwards anti-H immunoglobulin G was bound to protein A-beta-D-galactosidase to serve as the signal; 4-methylumbelliferyl-beta-D-galactoside was used as the substrate. The sensitivity threshold was 10(7) cells per ml. Direct EIA, indirect EIA, and pure-culture techniques were compared by using 48 samples of naturally contaminated foods and feeds. The direct EIA was more sensitive than the indirect EIA or pure-culture technique. Food samples were analyzed within 3 working days, and 32 samples were tested simultaneously in a single 96-well microtitration plate. False-positive or false-negative results did not pose a problem. This direct EIA is sensitive, rapid, and amenable to automation.     

R-ELISA: repeated use of antigen-coated plates for ELISA and its application for testing of antibodies to HIV and other pathogens.

In this paper we report on the evaluation of several procedures that allow for the repeated use of an antigen-coated, enzyme-linked immunosorbent assay (ELISA) plate for enzyme immunoassay (EIA). We have shown that antigen-coated ELISA plates that were incubated once with an aqueous solution containing 8 M urea, 2% sodium dodecyl sulfate and 2% mercaptoethanol, after an EIA, can be reused again for EIA without loss of antigenic capacity. Thus, in this procedure, after an EIA, the ELISA plates were washed once with the above solution and then in a buffer containing 20 mM Tris-HCl, pH 7.5, 0.1% Tween 20 and 500 mM NaCl. This washing protocol was shown to remove the primary antibody, enzyme-conjugated secondary antibody and substrate without removing the antigen from the ELISA plate microwells. Thus, an antigen-coated ELISA plate previously used for an assay could be reused. We tested this repeat ELISA (R-ELISA) procedure on high antigen-binding ELISA plates coated with two different plant virus proteins, a synthetic peptide, the p25/24 gag and the gp120 proteins of the human immuno-deficiency virus, or the staphylococcus enterotoxin protein. In each case tested, the procedure allowed for the repeated use of the same antigen-coated plates for EIA of the respective antibodies. This procedure should prove to be particularly valuable for mass screening of samples tested for HIV and other disease-causing agents.     

The use of microdot immunoenzyme analysis with visual detection for the determination of tularemia antibodies

The possibility of using the micropoint enzyme immunoassay (EIA) on a nitrocellulose membrane with the visual evaluation of results for the detection of tularemia IgG antibodies in hamadryas baboons at the postvaccinal period has been studied. The sensitivity of this assay has been compared with that of the passive hemagglutination (PHA) test, the microagglutination (MA) test and EIA with the spectrophotometric evaluation of results in plates. As shown in this study, EIA in the above-mentioned modification can be successfully used for the detection of tularemia antibodies in the blood serum. The sensitivity of micropoint EIA has proved to be not inferior to that of EIA in plates, while exceeding the sensitivity of the PHA test 10- to 20-fold and the sensitivity of the MA test 10- to 1,000-fold. This method is simple, reliable, highly sensitive, economic and requires no special equipment, which makes it highly promising for the diagnosis of tularemia and the evaluation of humoral immunity at the postvaccinal period.     

Production of monoclonal antibodies against the enzyme nuclease fromSerratia marcescens and evaluation of the individual antibodies for their use in EIA and in affinity chromatography

Summary Seven different monoclonal antibodies against the enzyme nuclease were compared with respect to their binding strength and to elution from EIA plates coated with antigen by ten different desorbing reagents. The combination of affinity ranking, obtained by specific titers, and results from desorption with chaotropic reagents and MgCl₂ serve as criteria for classifying the antibodies for use in EIA assays or for affinity chromatography. The introduction of an elution assay early during the screening of new hybridomas can lead to an early evaluation of parameters which are important for the subsequent use of monoclonal antibodies. Such a procedure will therefore save both costs and efforts.     

Salmonella detection in foods and feeds in 27 hours by an enzyme immunoassay

A heterogeneous enzyme immunoassay (EIA), which could be completed within 27 h, was developed for the detection of salmonellae in foods. Samples were subjected to the usual non-selective enrichment for 16–18 at 35°C and to a short (6 h) post-enrichment in a moderately selective broth. The EIA was carried out on polystyrene microtitration plates. A pooled polyvalent Salmonella flagellar antiserum and a protein A-alkaline phosphatase conjugate were used. The sensitivity of the enzyme immunoassay compared favorably with that of the conventional cultural technique for detection of Salmonella in 40 naturally contaminated food and feed samples. No sample was positive only by the cultural technique; samples positive only by the enzyme immunoassay were observed for feeds. Some specimens yielded high background values indicating possible interference from food proteins.     

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.     

紫外线辐照聚苯乙烯微孔板用于酶免疫测定的研究与表征

以重组人钙调素（rhCaM）、亲环素（rhCyP）、心磷脂和双链DNA（dsDNA）为包被抗原,建立了检测针对上述4种抗原自身抗体的间接ELISA方法,并对聚苯乙烯微孔板（PS）紫外线（UV）辐照前后进行了对比研究.结果发现：PS板经UV-辐照后,可显著改善酶免疫分析的测定效果,自身抗体的测定敏感度和重复性均有显著提高.原子力学显微镜（AFM）表征结果则提供了改善酶免疫分析的直接证据,抗原分子均匀平铺于UV-辐照的PS基底表面,而未经辐照的PS板则抗原分子的吸附率低,且分布不均并有成团聚集现象.X射线光电子能谱（XPS）分析表明,PS板经UV-辐照后,基底表面发生了氧化并引入了含氧的活性基团,O/C元素比较辐照前提高了6.9倍,改善了对抗原生物分子的亲水和化学反应性能,此亦即UV-辐照PS板改善对抗原分子固定效果的主要原因.     

The standardization of a 'spot-test' ELISA for the rapid screening of sera and hybridoma cell products II. The determination of binding capacity, binding ratio and coefficient of variation of different ELISA plates in sandwich and indirect ELISA

The different commercially available enzyme-linked immunosorbent assay (ELISA) plates were compared for their binding capacity for purified foot-and-mouth disease virus antigen or IgG, their binding ratio (a measure of the efficiency with which positive and negative serum samples may be distinguished), and their coefficients of variation within a plate, between plates and between batches of plates. No one plate could be described as having ideal characteristics, and the choice of ELISA plate depends on the use to which the ELISA is being put. For our purposes, viz. a 'spot-test' which rapidly and efficiently detects specific antibody when the levels of that antibody are low (hybridoma culture supernatants) or when the antibody is contaminated with other 'interfering' proteins (high concentrations of serum), we found that most of the PVC plates and, of the polystyrene plates, the Nunc Immunoplate I and Dynatech M129B plates performed well. The lowest coefficients of variation were obtained using Nunc Immunoplate I, Dynatech M129B and Falcon 3912 plates.     

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.     

Characteristics of antitoxic and antibacterial immune response in nontoxic forms of oropharyngeal diphtheria

The enzyme immunoassay system (EIA) for differentiation of antibodies in therapeutic heterogeneous antitoxic serum and antibodies to Corynebacterium diphtheriae toxigenic strains in patients and carriers was developed. The use of EIA permitted the dynamic evaluation of the characteristics of humoral antitoxic and antibacterial immune response in 50 patients with the localized and disseminated forms of stomatopharyngeal diphtheria and 14 "healthy" carriers of toxigenic C. diphtheriae. As revealed in this study, the symptoms of the disease in patients with disseminated forms of stomatopharyngeal diphtheria developed in the presence of statistically significant low quantitative values of antitoxic and antibacterial antibodies to C. diphtheriae antigens. In the group of patients with the localized forms of the disease the initially low level of antitoxic antibodies was detected with the concentration of antibacterial antibodies remaining unchanged. During the period of convalescence the levels of antitoxic antibodies in both groups reached those of healthy persons. In case of localized forms of the disease the level of antibacterial antibodies decreased as compared with healthy persons, starting from the second week of the disease. The period of convalescence in the disseminated forms was characterized by the low concentration of antibacterial antibodies. Carrier state was formed in the presence of high levels of antitoxic antibodies and significantly low levels of antibacterial ones.     

What criteria should be used to select biodiversity indicators?

The conservation of biodiversity is a major goal in nature conservation, but measuring the total biodiversity of a site or a region is not possible; thus there is a great demand for indicators to represent biodiversity. To be able to make use of indicators, criteria must first be established for their selection, and the degree to which the indicators meet the criteria must be tested. However, the purposes for which indicators are applied—and thus sometimes the criteria themselves—differ between ecological science and environmental policy. As transparency in choosing and testing suitable biodiversity indicators will optimize the results of an indicator, this article first aims to determine if there are common approaches in selecting biodiversity indicators in ecology and environmental policy. Second, we asked which criteria biodiversity indicators were scientifically tested against to determine their suitability. To answer these questions, we analyzed papers on biodiversity indicators referenced in the Web of Science. Our results demonstrate different patterns for selecting biodiversity indicators in the different fields of application. In ecology, the quality of indicators is mainly determined by a close relationship between indicator and indicandum (i.e., indicated phenomenon), while the relevance of an indicator for a given issue, e.g., reserve selection or an assessment of a certain impact, is of paramount importance for conservation policy. Surprisingly, few biodiversity indicators are empirically tested to determine if they meet the criteria by which they were purportedly chosen. We argue that this is due to the different conceptualizations of biodiversity indicators in science and environmental policy. Since the suitability of biodiversity indicators remains untested in many cases, our findings suggest room to make better use of indicators in ecology and environmental policy. As the results of ecological research are put to use to solve environmental problems, the selection of indicators for ecological research should correspond to a large extent with those used in environmental policy. Further, to assess the suitability of a biodiversity indicator, it should be tested against all of the criteria relevant for its selection.