Diagnostic test system for detecting the meningococcal antigen by erythro-immunoadsorption

The authors have developed a test-system for detection of group A meningococcal polysaccharide, based on the sandwich erythro-immunoadsorption ultra-microtechnique with the use of the Terasaki plates as a solid-phase carrier. The system, equivalent to ELISA in its high sensitivity and specificity, is more rapid and less expensive, permitting the detection of 1 mg/ml of the antigen in 20 microliter of the tested liquid within an hour. The results of the study of CSF samples from 28 patients with meningococcal infection were in good correlation with the ELISA results. The new test-system is recommended for practical use as a routine technique for the specific diagnosis of meningococcal meningitis and for the control of the effectiveness of the treatment.     

Use of recombinant HBcore antigen for development of the diagnostic test-system

The plasmid construction expressing recombinant HBc antigen (HbcAg) in Escherichia coli cells under the control of the PL promoter of phage I, was obtained. The specific activity of the antigen thus obtained was controlled by the enzyme immunoassay (EIA) method and compared with the reference system "AxSYM CORE assay" ("Abbott", USA) with four panels of sera (altogether 111 samples). The coincidence of the results of the compared test system with the reference was 96.4%, which made it possible to recommend this genetic construction of recombinant HBcAg for the production of EIA systems.     

The labeled antigen method of immunoenzymatic staining.

A two stage immunohistological technique (the "labeled antigen" procedure) has been assessed for the detection of a variety of human and animal cytoplasmic constituents in tissue sections. In this method specific antiserum is followed by antigen complexed to horseradish peroxidase or to alkaline phosphatase. The primary antibody acts bivalently, linking the labeled antigen to antigen in the tissue section. The major advantage of this technique is that nonantigen specific antibody in the primary antiserum cannot cause nonspecific staining since it has no affinity for the antigen:enzyme complex. Consequently the specificity of the reaction is assured, background staining is minimized and the total staining time (from wax section to mounted slide) can be reduced to as little as 30 min. Further advantages include the possibility of labeling Ig allotypes and the high efficiency of enzyme utilization. Covalent human IgG:horseradish peroxidase complexes can also be used in a triple sandwich in conjunction with human anti-viral or autoimmune antibodies.     

Double immunoenzymatic staining for the simultaneous detection of Epstein-Barr virus induced antigens

A double indirect immunoenzymatic staining was developed for the simultaneous visualization of Epstein-Barr virus-induced early antigens and virus capsid antigens in P3HR1 lymphoblastoid cell line. The double immunocytochemical staining was performed with a four-stage and a two-stage procedure employing human sera and monoclonal antibodies against Epstein-Barr virus-induced antigens, followed by the addition of specific alkaline phosphatase and peroxidase labeled antisera. The selection of substrates yielding reaction products of contrasting colours enabled the observer to distinguish cells expressing Epstein-Barr virus capsid antigens (blue) from cells expressing Epstein-Barr virus early antigens (brown).     

Double immunoenzymatic staining for the simultaneous detection of Epstein-Barr virus induced antigens

Summary A double indirect immunoenzymatic staining was developed for the simultaneous visualization of Epstein-Barr virus-induced early antigens and virus capsid antigens in P3HR1 lymphoblastoid cell line.The double immunocytochemical staining was performed with a four-stage and a two-stage procedure employing human sera and monoclonal antibodies against Epstein-Barr virus-induced antigens, followed by the addition of specific alkaline phosphatase and peroxidase labeled antisera.The selection of substrates yielding reaction products of contrasting colours enabled the observer to distinguish cells expressing Epstein-Barr virus capsid antigens (blue) from cells expressing Epstein-Barr virus early antigens (brown).     

Adaptation of immunoenzymatic method for detection of antibody coated bacteria in urine

The modified immunoenzymatic method for detection of antibody coated bacteria (IP ACB) was compared with immunofluorescence technique (IF ACB) in the diagnosis of urinary tract infection. For the study 100 patients were employed with significant and insignificant bacteriuria. It was found that 81% of the results obtained by IP ACB and IF ACB were identical, however the immunoenzymatic method was more sensitive than immunofluorescence. Moreover, the IP ACB technique is simpler, less time consuming and may be performed by using the ordinary optic microscope.     

Comparison of the radioimmunological and immunoenzymatic methods in detection of HBsAg]

We have compared the solid-phase radioimmunoassay(SPRIA) with a solid-phase enzyme-immunoassay (EIA) in the detection of hepatitis B surface antigen (HBsAg). 708 sera from blood donors and 500 sera from patients with various diseases (acute and chronic hepatitis, chronic renal failure in hemodialytic treatment) were tested for HBsAg with both methods. 208 sera (17,2%) were found to be positive in SPRIA and 209 sera (17,3%) in EIA. Two HBsAg positive sera were tested in dilution series with both methods, too. The results show that the sensitivity and specificity of the EIA compare very favourably with those of the SPRIA.     

The phenomenon of HLA antigen modification in meningococcal infection

212 patients with the generalized forms of meningococcal infection have been examined. The intensity and quality of lymphocytotoxic reaction in the acute phase and the convalescence period of the disease have been analyzed and the blank calculation has been made at different clinical forms of meningococcal infection. The formation of modified tissue antigens at the acute stage of the disease in the presence of intoxication is shown and the probable role of the intensity of bacteremia in this formation is discussed.     

Phylogenetic evidence for frequent positive selection and recombination in the meningococcal surface antigen PorB

Previous estimates of rates of synonymous (d(S)) and nonsynonymous (d(N)) substitution among Neisseria meningitidis gene sequences suggested that the surface loops of the variable outer membrane protein PorB were under only weak selection pressure from the host immune response. These findings were consistent with studies indicating that PorB variants were not always protective in immunological and microbiological assays and questioned the suitability of this protein as a vaccine component. PorB, which is expressed at high levels on the surface of the meningococcus, has been implicated in mechanisms of pathogenesis and has also been used as a typing target in epidemiological investigations. In this work, using more precise estimates of selection pressures and recombination rates, we have shown that some residues in the surface loops of PorB are under very strong positive selection, as great as that observed in human immunodeficiency virus-1 surface glycoproteins, whereas amino acids within the loops and the membrane-spanning regions of the protein are under purifying selection, presumably because of structural constraints. Congruence tests showed that recombination occurred at a rate that was not sufficient to erase all phylogenetic similarity and did not greatly bias selection analysis. Homology models of PorB structure indicated that many strongly selected sites encoded residues that were predicted to be exposed to host immune responses, implying that this protein is under strong immune selection and requires further examination as a potential vaccine candidate. These data show that phylogenetic inference can be used to complement immunological and biochemical data in the choice of vaccine candidates.     

Structural variation and immune recognition of the P1.2 subtype meningococcal antigen

Neisseria meningitidis is a globally important cause of bacterial meningitis and septicemia. No comprehensive antimeningococcal vaccine is available, largely as a consequence of the high sequence diversity of those surface proteins that could function as components of a vaccine. One such component is the protein PorA, a major surface porin of this Gram-negative organism that has been used in a number of experimental and licensed vaccines. Here we describe a series of experiments designed to investigate the consequences for antibody recognition of sequence diversity within a PorA antigen. The binding of a 14-residue peptide, corresponding to the P1.2 subtype antigen, to the MN16C13F4 monoclonal antibody was sensitive to mutation of five out of the six residues within the epitope sequence. The crystal structure of the antibody Fab fragment, determined in complex with the peptide antigen, shows a remarkably hydrophobic binding site and interactions between the antigen and antibody are dominated by apolar residues. Nine intrachain hydrogen bonds are formed within the antigen which maintain the beta-hairpin conformation of the peptide. These hydrogen bonds involve residues that are highly conserved amongst different P1.2 sequence variants, suggesting that some positions may be conserved for structural reasons in these highly polymorphic regions. The sensitivity of antibody recognition of the antigen towards mutation provides a structural explanation for the widespread sequence variation seen in different PorA sequences in this region. Single point mutations are sufficient to remove binding capability, providing a rationale for the manner in which different meningococcal PorA escape variants arise.     

An immunoenzymatic method for improving the sensitivity of antigen measurement by electroimmunodiffusion techniques. Application to the quantification of human alpha-fetoprotein.

A double antibody technique of electroimmunodiffusion, which uses glucose oxidase-labelled sheep antibodies to rabbit immunoglobulins as second antibody, is described. Primary antigen-antibody reaction is carried out with a rabbit antiserum by electroimmundiffusion. The glucose oxidase-labelled immunoreagent, being of general application, can serve for the quantification of different antigens and is here used for measurement of low levels of human alpha-fetoprotein. Reproducible results in the range of 50-800 ng/ml were obtained with a variation coefficient of 5 to 10%.     

Test-systems for immunoenzymatic determination of 6-deoxobrassinosteroids

26-Hydroxyderivatives of 6-deoxo-24-epicastasterone and 6-deoxo-28-norcastasterone were synthesized for preparation of the corresponding haptens and conjugates with horseradish peroxidase and bovine serum albumin. These preparations were further applied to a production of antisera. Test-systems for the enzyme immunoassay of phytohormones of the 6-deoxobrassinosteroid group were elaborated.     

Simultaneous detection of cytokine and immunophenotype at the single cell level by immunoenzymatic double staining

Summary The goal of this study was to establish a generally applicable immunoenzymatic method for the simultaneous detection of cytokine and immunophenotype at the single cell level. Evaluating various cell preparations and staining protocols, we found that permeabilization by saponin (0.1%) is very efficient, in combination with glutaraldehyde (0.04%) as fixative. Among various staining procedures, sequential immunoperoxidase labelling of the cytokine by use of diaminobenzidine, and detection of the immunophenotype by use of 4-chloronaphthol proved most discriminative. The typical localization of the cytokine reaction product (Golgi staining) within the cell, and the ringlike staining for the immunophenotype on the cell surface, allowed precise identification of double-labelled cells. Primary monoclonal antibodies from the same species could be used without loss of sensitivity and specificity for either or both antigens. This method thus provides the opportunity to study morphology, cytokine and immunophenotype simultaneously at the single cell level with standard equipment. Its application for the analysis of tissue samples is in progress, and may allow us to incorporate the cytokine-type as a new parameter in histopathological diagnostics.     

Obtaining erythrocyte diagnosticum for the detection of chlamydial antigen

It is shown possible to obtain erythrocyte diagnosticum for detection of chlamydial antigen, whose cell basis consists of a formalinized suspension of ram erythrocytes, sensibilized with hyperimmune antichlamydial sera by means of the amydol. High sensitivity and specificity of the diagnosticum, absolute correlation with the data obtained in the complex examination of patients with the urogenital tract pathology of the chlamydial etiology by other methods were determined in the course of investigation in the indirect hemagglutination test with diagnosticums of scrape specimens from these patients.     

Fast-in Situ hybridization and immunoenzymatic color pigment detection of mouse bone marrow micronucleus

The development of a whole mouse genomic DNA probe coupled to color pigment painting detection methodology can accurately verify mouse micronuclei induced by chemicals or drugs leading to a lower probability of potential artifacts. Using color pigment painting detection of probes in conjunction with Wright's Giemsa counterstain instead of the current fluorescence detection technology ensures low cost, high resolution permanent documentation of slides for a particular test compound. The permanent color pigment-detected micronuclei and adjoining counterstain allows slides to be stored for future analysis without enhancing the signal or adding antifading agents that are associated with fluorescence detection. Combining innovative technology such as fast-in situ hybridization of DNA probes with immunoenzymatic color pigment detection provides rapid verification of true micronuclei (DNA containing) within 2-3 hr.     

Diagnostic suspension for detection of the infective agent of tularemia

The matrix for obtaining silica-based diagnosticum was selected, its activation with surfactant was optimized and its immobilization with tularemia immunoglobulins was carried out. In the glass suspension agglutination test (SAT) the sensitivity of the diagnosticum was 3.125 x 10(6) to 6.25 x 10(6) microbial cells per ml; the duration of SAT, including the evaluation of its results, was 1-5 minutes.     

Reliability of the detection of meningococcal gamma-glutamyl transpeptidase as an identification marker for Neisseria meningitidis

Detection of gamma-glutamyl transpeptidase (GGT; ggt ) activity is one of the useful methods for a specific identification of Neisseria meningitidis. However, we previously happened to isolate a ggt -deficient N. meningitidis strain (NIID113) from a healthy carrier. In this study, in order to re-examine the reliability of the marker, we again investigated the GGT activity of 245 N. meningitidis human isolates and identified two other GGT-defective N. meningitidis isolates besides NIID113. The isolation frequency (1.2%) of ggt mutants among human isolates strongly confirmed the 98.8% reliability of GGT activity as the identification marker for N. meningitidis.     

Bacterial test-system for a primary screening of the substances with potential anti-cancer activity

A principle possibility of antitumour activity test in bacterial system represented by Escherichia coil of the wild type and its MS2-induced mutant has been shown. The initial bacterial strain is an indicator of toxic properties of the tested substances and the mutant one is a specific test-culture modelling a tumour cell. The comparison of the data described for eucaryotes with the data obtained using the proposed bacterial test system confirms an adequate response of both strains to the substances with the proved antitumour properties. The data are considered as very promising for the further improvement of this test system towards its application for primary screening of antitumour substances.