HEMOGLOBIN AS A CAPTURE AGENT FOR LIPOPOLYSACCHARIDE ANTIGENS IN THE ENZYME IMMUNOASSAY OF GRAM NEGATIVE BACTERIA

Abstract The affinity of hemoglobin for lipopolysaccharides (LPS) was exploited in its use as an inexpensive capture agent for LPS antigens in the enzyme immunoassay (EIA) of Gram negative bacteria. Two EIA formats were examined. In one, the macroporous solid phase Polymacron ^TM coated with hemoglobin was used to capture cholate-heat extracted LPS antigens from broth cultures of representative Gram negative bacteria, including different Salmonella serotypes, which were then detected immunoenzymatically using specific antibodies. This provided a rapid, simple and inexpensive dot blot assay for these bacteria which minimized the requirement for laboratory equipment. In another format, a microtiter plate EIA was developed in which cholate-heat extracted Salmonella . LPS antigens were captured in hemoglobin-coated wells. The microtiter plate format is automatable and will therefore be useful in laboratories with high sample throughputs. While most of the results reported here focus on the detection of Salmonella spp., we also demonstrate the applicability of this system in the assay of Escherichia coli O157 LPS antigens .     

Early detection and rapid identification of Streptococcus pneumoniae and Haemophilus influenzae in acute pneumonias

The comparative study of the diagnostic value of the enzyme immunoassay (EIA), indirect immunofluorescence (IF) and countercurrent immunoelectrophoresis (CIE) was made. The serological identification of the isolated and reference pneumococci (19) and H. influenzae (38) strains revealed the possibility of using all three microanalytical methods for this purpose. The study of pneumococcal and H. influenzae antigens in native sputum obtained from 74 patients with acute pneumonia showed that EIA and indirect IF were highly sensitive, their sensitivity considerably exceeding that of the bacteriological analysis. Pneumococcal antigens were detected in 66.2% of patients by EIA and in 54.0% of patients by indirect IF, while H. influenzae antigens were detected in 58.1% of patients by EIA and in 67.6% of patients by indirect IF. The sensitivity of CIE proved to be considerably lower; in the detection of pneumococcal antigens it was level with the sensitivity of the bacteriological analysis (23.0%) and H. influenzae antigens could be detected only in 27.0% of patients.     

Elaboration of enzyme immunoassay based on recombinant antigens and intended for diagnostics of syphilis

Enzyme immunoassay (EIA) using recombinant antigens for the detection of Treponema pallidum-specific antibodies in sera of syphilis patients was developed. Four low-molar-mass Treponema antigens (Tp15, Tp17, TmpA, Tp47) were investigated; 17- and 47-kDa proteins were demonstrated as immunodominant as they permitted to obtain the most sensitive EIA. Using a mixture of these proteins a 3rd-generation-EIA kit Dia-Syph was constructed, its sensitivity being 99.4% during tests of 165 sera of syphilitic patients. No false result was obtained on the commercial panel PSS01 (BBI, USA). The specificity of the elaborated test system (99.7%) was determined on 295 sera.     

Development of a method for the quantitative determination of Bordetella pertussis toxin and the prospects of its use for diagnosis

Two variants of the enzyme immunoassay (EIA) systems for the determination of B. pertussis toxin (BPT), the "double sandwich" system and the competitive assay system, have been developed. For the titration of BPT in B. pertussis antigens the use of fetuin as the affinity base is preferable, and not antibodies from different paired animals. Of the two variants, the competitive EIA is more promising for diagnostic purposes.     

The reasons for false positive results with recombinant preparations in HIV infection]

Enzyme immunoassay (EIA) with the use of test systems based on recombinant HIV antigens has yielded positive results much more often than that with the test systems based on HIV lysate. In comparison with the use of protein A-peroxidase conjugate, the use of antiglobulin conjugates decreased the specificity of EIA, thus ruling out the false positive results. The tested sera containing antibodies to antigens of enteric bacilli could yield false positive EIA results with test systems prepared from recombinant antigens due to the interaction of these antibodies with antigens of enteric bacilli. A conclusion on the usefulness of the control for the presence of the components of enteric bacilli, producers of HIV antigen, and their elimination from recombinant preparations is made.     

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).     

Development of an immunoenzyme test system for determining the antigens of feed yeasts

An enzyme immunoassay (EIA) system for the detection of fodder yeast antigens in the air of production areas at fodder protein producing plants has been developed. The method has proved to be highly sensitive and specific and shows advantages in comparison with the nonspecific method of low sensitivity, currently used at such plants. The sensitivity of solid-phase EIA techniques is 0.001 micrograms/ml (for protein) or 10(2)-10(3) cells/ml, and 10 ng/ml for soluble antigen. No cross reactions with bakers' yeast antigen have been observed.     

An immunoenzyme system for determining antibodies to Streptococcus pneumoniae polysaccharides in biological fluids

The enzyme immunoassay (EIA) system for the determination of antibodies to capsular polysaccharides of pneumococci, serotypes 1, 3, 6B, 8, 9N, 15F, 23F, and C-polysaccharide has been developed on the basis of poly-L-lysin-modified antigens. The use of isotype-specific conjugates in this system permits the detection of IgG and IgA antibodies in different biological fluids: blood serum, pleural fluid, saliva, milk. Samples obtained from children with pneumococcal infection and from nursing mothers have been studied. As shown in this study, the EIA system can be used for the evaluation of the dynamics of pneumococcal infection in children.     

清洁级实验动物四种病毒抗体玻片酶免疫检测试剂盒的研制

本文报道对清洁级实验动物应排除的四种病毒（淋巴细胞脉络丛脑膜炎病毒、小鼠脱脚病病毒、鼠肝炎病毒和仙台病毒）抗体玻片酶免疫（ＥＩＡ）检测试剂盒的研制。四种病毒感染的细胞和对照细胞经冷丙酮固定于载玻片上制成特异性抗原和对照抗原,此四种病毒的抗血清各１０份和ＳＰＦ小鼠血清２０份分别与四种病毒的特异性抗原和对照抗原进行ＥＩＡ交叉试验,结果显示,抗原只与其相应抗血清发生特异性显色反应,与非特异性小鼠血清和ＳＰＦ小鼠血清不显色。与ＨＩ或ＥＬＩＳＡ方法比较,通过对１１２份普通小鼠血清进行测试,结果表明,ＥＩＡ对仙台病毒抗体的检出率（１９．６％）显著高于（＜０．００５）ＨＩ（６．３％）,对小鼠脱脚病病毒抗体的检出率（２３．３％）与ＨＩ（２１．４％）无显著性差异（Ｐ＞０．０５）。ＥＩＡ对淋巴细胞脉络丛脑膜炎病毒和鼠肝炎病毒抗体的检出率分别为１．８％和７１．２％,ＥＬＩＳＡ对两种病毒抗体的检出率分别为１．８％和６７．６％,两种方法对两种病毒抗体的检出率无显著性差异（Ｐ＞０．０５）。重复性试验表明两批四种病毒抗体试剂盒对１０８份小鼠血清两次测定的符合率为９６～１００％。四种病毒的ＥＩＡ抗原在－１８℃保存１２个月或在２－８℃保存３     

Comparative evaluation of two commercially available antigen enzyme immunoassays (EIA) for the detection of Legionella pneumophila urinary antigen in frozen non-concentrated urine samples

We evaluated two commercial enzyme immunoassay kits, Binax EIA (for detection of soluble antigen of Legionella pneumophila serogroup 1) and Biotest EIA (for detection of antigens of Legionella pneumophila serogroups and other Legionella spp.) in order to introduce this test routinely for the diagnosis of Legionnaires' disease (LD) in our Laboratory. Frozen non-concentrated urine samples belonging to 45 patients with and without LD were tested. The sensitivity of Binax EIA and Biotest EIA was 47.4% and 42.1% respectively, the specificity was 95% by both tests. Biotest did not detect antigen from a patient with culture-proven infection of L. pneumophila serogroup 6. The detection of urinary antigen by both EIA tests is a useful tool for rapid diagnosis of LD, especially when samples are unavailable for culture; the sensitivity may be increased if the assay is performed on unfrozen and concentrated samples.     

Diagnostic value of different laboratory methods in the diagnosis of pneumonia caused by Haemophilus influenzae type B

Comparative analysis of the diagnostic value of different laboratory methods in the diagnosis of H. influenzae b (Hib) pneumonia in children (bacteriological method, latex agglutination, counter immunoelectrophoresis, the passive hemagglutination test and the enzyme immunoassay (EIA) was carried out. EIA proved to be the most informative method for the diagnosing Hib pneumonia. EIA makes it possible to detect specific Hib antigens in different clinical materials in 48.8% of cases, as well as high titers of antibodies to mis infective agent in 61.7% of cases. The authors propose the unified criteria of the laboratory diagnosis of Hib infection in children.     

Beta-galactosidase from E. coli as a marker in immunoenzyme analysis

In the present review the authors analyze factors influencing sensitivity of the enzyme immunoassay (EIA). beta-Galactosidase from E. coli was chosen as a marker enzyme. Physico-chemical properties of the enzyme, detection methods and various ways of obtaining chemical conjugates with antigens and antibodies are discussed. Some examples of using beta-galactosidase as a label in homogeneous and heterogeneous EIA are given which enables different compounds to be analyzed with a high sensitivity. New approaches employing gene engineering for constructing "fusions" between beta-galactosidase and antigens (alternative to chemical conjugates) are discussed that can be used in the near future.     

Immunoenzymatic detection of specific brain antigens as a criterion of the permeation of blood-brain barrier in rats with acute cerebral ischemia

EIA detection system for the measurement of alpha 2 M globulin and GFAP antigen has been developed. The limit of the sensibility was only 1 ng/ml for alpha 2M and 0.8 ng/ml for GFAP. The system was used for the studies of the penetration through the blood-brain barrier in rats with experimental acute brain ischemia. The measurement of alpha 2M and GFAP antigens by EIA technique 16-20 hours after the occlusion of the carotid artery has revealed disturbances in the blood-brain barrier permeability for specific brain proteins. The method is recommended for indirect evaluation of the blood-brain barrier functional disorders.     

Use of microcapsular leptospiral antigens for detecting specific antibodies

The sensitivity of microcapsular leptospiral antigens, produced by Japan Lyophilization Laboratory and intended for use in tests for the detection of antibodies to leptospires in the sera of experimentally immunized laboratory animals, were studied. The comparative study of the microcapsular agglutination (MCA) test and other serological tests, such as the microagglutination (MA) test and the indirect enzyme immunoassay (EIA), was made. The leptospiral antigens under study were found to actively react with serospecific and group-specific antibodies. In infected guinea pigs and rabbits specific antibodies could be detected from days 3-4 in the MCA test and only from days 5-7 in the MA test. The average antibody level determined by titration in the MCA test was 3.3 times higher and in indirect EIA, 4.3 times higher than that determined by titration in the MA test. These data make it possible to recommend the use of microcapsular leptospiral antigens for the early diagnosis of leptospirosis.     

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.     

An immunoenzyme method of isolation of foot-and-mouth disease virus by using beta-lactamase conjugate with virus-specific antibodies

It was shown that in was feasible to use conjugates of virus-specific antibodies and beta-lactamase from Bacillus licheniformis 749/c to identify aphthosa virus antigens. The antigen titers determined by enzyme immunoassay (EIA) using a beta-lactamase conjugate were 5-64 times higher than the analogous indices of the complement fixation test. Unlike EIA, that by using the antibody conjugates with peroxidase or alkaline phosphatase there were observed no "background" responses.     

Autoantibody biomarker opens a new gateway for cancer diagnosis

The list of cancer markers of current interest has grown considerably, but none of the markers used in clinical work is a true tumor marker. These cancer biomarkers are based on the determination of tumor antigens. Here, we report a single method of autoantibody enzyme immunoassay (EIA) screens for a spectrum of serum tumor markers. A comparison of the autoantibody-based EIA to conventional antigen EIA kits, using receiver operating characteristic (ROC) plots, showed that the autoantibody EIA can significantly enhance the sensitivity and specificity of tumor markers. The detection of serum autoantibodies for a spectrum of serum tumor markers, as demonstrated here, suggests that most, if not all, serum cancer biomarkers produce autoantibodies. A unique autoantibody biomarker screening method, as presented here, might therefore facilitate achieving the accurate and early diagnosis of cancer.     

Antigens of Coccidioides posadasii as an Important Tool for the Immunodiagnosis of Coccidioidomycosis

Serologic diagnosis has been presented as a safe alternative for coccidioidomycosis. However, commercial kits based on coccidioidal antibodies available in the USA are considered too expensive for laboratories outside that country. In this study, we describe the preparation of antigens for detection of human coccidioidal antibodies by the immunodiffusion test (ID) and enzyme immunoassay (EIA). Antigens were tested against serum samples from patients with coccidioidomycosis, histoplasmosis and paracoccidioidomycosis, as well as healthy individuals. The highest reactivity in the ID tests was seen in the F0-90 antigen. In the EIAs, the best results were obtained with the F60-90 antigen. None of the serum samples from healthy individuals were recognized by any of the antigen extracts tested by ID or EIA. In conclusion, the F0-90 and F60-90 antigens have the potential to be commercially employed in presumptive diagnosis of coccidioidomycosis by ID or EIA, respectively. The tests could improve serological diagnosis of coccidioidomycosis in South America.     

The optimization of immunoenzyme analysis with conjugates of virus-specific antibodies and alkaline phosphatase

The data obtained in the study of the dependence of sensitivity of enzyme immunoassay (EIA) on chromo- and fluorogenic substrates used in this assay are presented. The sandwich variant of EIA, carried out with the use of antibodies labeled with alkaline phosphatase, has been shown to be 4-170 times, sometimes 500 times, more sensitive (in terms of concentrations at which aphthous fever virus antigens can be detected) than the complement fixation test and 1.8-64 times more sensitive than the passive hemagglutination test.     

Evaluation of OIDx Histoplasma Urinary Antigen EIA

Mycopathologia - A sandwich enzyme immunoassay (EIA) for the detection of Histoplasma antigens (Ag) in urine, developed by Optimum Imaging Diagnostics (OIDx) was evaluated. A verification using a...