Immunoenzyme method in the diagnosis of meningococcal infections

The materials on the development and use of the test system, based on the enzyme-linked immunosorbent assay (ELISA) and intended for the detection of specific group A and C meningococcal polysaccharides and type b Haemophilus influenzae polysaccharide in the spinal fluid of patients, are presented. In this work commercial preparations manufactured in the USSR were used, and all parameters of the assay were developed on the basis of these preparations. The study was made on the samples of spinal fluid from 410 patients; of these, 203 had meningococcal infection, 57 had purulent bacterial meningitides and 150 had other diseases (acute respiratory diseases, influenza, etc.). As demonstrated by the results of this study, ELISA proved to be a highly specific and sensitive technique. In the investigation of the spinal fluid samples from the patients with meningococcal infection the use of ELISA with bacteriological techniques increased the number of positive results to 67%; with countercurrent electrophoresis, to 78%; and with bacterioscopy, to 83.8%. ELISA is recommended for practical use as an auxiliary laboratory technique and as a rapid method for the diagnosis of meningococcal infection.     

Preparations for serodiagnosis of diseases due to causative agents of ixodes tick-borne borreliosis (Lyme disease). Communication 3. Comparative study of enzyme immunoassay test-systems for detection of IgM to Borrelia burgdorferi sensu lato]

Three foreign and one Russian ELISA test-systems for detection of IgM to Borrelia burgdorferi sensu lato were comparatively studied with the use of the clinical material in an encoded experiment. In the foreign ELISA test-systems based on the use of native antigens of borrelia, cross reactions with sera from patients with syphilis, Epstein-Barr infection, cytomegalovirus infection and systemic lupus erythematosus were detected. The Russian recombinant ELISA test-system Borreliosis-ELISA-IgM showed high sensitivity and specificity. The simultaneous use of the test-systems for detecting IgG and IgM significantly increased the efficacy of diagnosis of early borreliosis.     

Enzyme-linked immunosorbent assay of chlorampenicol in foodstuff

A test-system based on enzyme-linked immunosorbent assay (ELISA) for the quantitative detection of chloramphenicol (CAP) in foodstuff has been developed. The detection limit of the method was 0.05 μg/l. The procedures for milk samples preparation of various fat content and chicken muscles were optimized. Before the analysis milk was diluted 5-fold with a buffer. The detection limit for milk was 0.3 μg/l; recoveries varied from 74 to 118%. Two protocols for chicken muscles preparation were elaborated; extraction with buffer (the express method) and extraction with acetonitrile. The detection limits of CAP in chicken muscles were 0.5 and 0.3 μg/kg, respectively; recovery values were 71–107% and 95–115%, respectively. The results of residual amounts of CAP detection in foodstuff by ELISA and HPLC-MS were in good correlation.     

Increased specificity of immunoenzyme analysis for diagnosing meningococcal infection

The ELISA test system for the detection of polysaccharide antigens of meningococci, groups A and C, on the basis of the neutralization of specific antibodies has been developed. The specificity of this reaction is determined by the chemically pure preparations of group A and C meningococcal polysaccharides. The sensitivity of this test system based on the neutralization of antibodies is not inferior to that of ELISA with the use of double antiserum.     

Diagnostic test-system for the immunoenzymatic detection of meningococcal antigen

To detect meningococcal antigen, the use of the enzyme-labeled immunosorbent assay (ELISA), a new variant of the immunoenzymatic method, permitting one to carry out quantitative analysis, is proposed. The optimum conditions for the test to detect group A meningococcal antigen, as well as the procedure for the approbation of the test on patients with meningococcal infection and on healthy persons, have been worked out. The method is shown to be highly specific and sensitive.     

Use of recombinant proteins carrying antigenic determinants of the envelope proteins of AIDS virus in the diagnosis of acquired immunodeficiency syndrome

The testing of sera from patients with AIDS and AIDS-related complex, using ELISA with recombinant env protein produced by E. coli, gave a 94% coincidence with the results obtained on "Organon" test-system. Sera samples that gave different results in two tests lacked antibodies to env-encoded proteins, as revealed by immunoblotting assay. The application of the above test for the estimation of the disease prognosis is discussed.     

False positive diagnosis of meningococcal infection by the IS1106 PCR ELISA

At a time when optimal case ascertainment for meningococcal infection is a high priority, the need for non-culture case confirmation, in particular by DNA amplification, is seen as being of vital importance to assist contact management and cluster recognition. A solution hybridisation assay with colorimetric microtitre plate detection (polymerase chain reaction-enzyme-linked immunosorbent assay (PCR ELISA)) has been developed using the multicopy insertion sequence IS1106 which had reportedly achieved a specificity of 100% and was described as being meningococcal specific. This PCR ELISA assay was evaluated on specimens from over 5000 patients at the national Meningococcal Reference Unit (MRU) between late 1995 and early 1997 and was found to be highly sensitive. Insertion sequences, however, are genetically mobile with the ability to spread between species and even genera. During the evaluation period of the IS1106 PCR ELISA a number of false positives proved to be caused by organisms other than N. meningitidis were recorded resulting in the withdrawal of this assay as a front line screening assay for routine confirmation of meningococcal infection.     

Evaluation of the Applied Biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA

In a period where the proportion of culture confirmed cases in the UK has been steadily declining, diagnosis by PCR has been used to increase the number of confirmed cases and provide additional epidemiological data. This report presents a comparative evaluation of the fluorogenic probe-based 5' exonuclease assay (Taqman) using the Perkin-Elmer Applied Biosystems automated sequence detection system 7700 with previously reported polymerase chain reaction enzyme-linked immunosorbent (PCR ELISA) assays for the detection of meningococcal DNA in CSF, plasma and serum samples. Taqman assays developed were based on the detection of a meningococcal capsular transfer gene (ctrA), the insertion sequence IS1106 and the sialytransferase gene (siaD) for serogroup B and C determination and compared with similar assays in a PCR ELISA format. The Taqman ctrA assay was specific for Neisseria meningitidis, however the IS1106 assay gave false positive reactions with a number of non-meningococcal isolates. Sensitivity of the Taqman ctrA, IS1106 and siaD assays testing samples from culture-confirmed cases were 64, 69 and 50%, respectively, compared with 26, 67 and 43% for the corresponding PCR ELISA assays. Improvements to the DNA extraction procedure has increased the sensitivity to 93 and 91% for the TaqMan ctrA and siaD assays, respectively, compared to culture confirmed cases. Since the introduction of Taqman PCR a 56% increase in laboratory confirmed cases of meningococcal disease has been observed compared to culture only confirmed cases. The developed Taqman assays for the diagnosis of meningococcal disease enables a high throughput, rapid turnaround of samples with considerable reduced risk of contamination.     

A novel monoclonal antibody to Neisseria meningitidis serogroup X capsular polysaccharide and its potential use in quantitation of meningococcal vaccines

A novel murine hybridoma monoclonal antibody (MAb) was produced against the capsular polysaccharide (CP) of Neisseria meningitidis serogroup X (MenX) in order to develop a sandwich enzyme linked immunosorbent assay (ELISA) for the quantitation of the meningococcal polysaccharide. The MAb only reacted with the CP from MenX and did not react with CPs from N. meningitidis serogroups A, C, Y and W (MenA, MenC, MenY, MenW). The affinity constant (Ka) of the MAb measured by non-competitive ELISA was 7.25 × 10⁷ M⁻¹. The application of this MAb in a sandwich ELISA was demonstrated by its ability to properly quantitate three lots of an experimental meningococcal CP-based vaccine. The MAb obtained in this work could be a valuable reagent for the detection and quantitation of future meningococcal vaccines containing MenX CP.     

检测四价脑膜炎球菌多糖疫苗中A群多糖含量ELISA法的建立

目的建立双抗体夹心ELISA法,对A群流脑多糖抗原进行特异性定量测定。方法制备抗A群多糖的特异性多克隆抗体,所得抗血清经辛酸-硫酸铵沉淀法纯化后,用过碘酸钠法制备辣根过氧化物酶标记多克隆抗体。分别以抗A群多糖多克隆抗体作为包被抗体及酶标二抗,建立双抗体夹心ELISA法,优化反应条件,对A群多糖抗原进行特异性定量测定。结果一系列验证试验表明,该法特异性较好,未检出与C、Y、W135群多糖的交叉反应;1.25～20 ng/mL多糖浓度范围的剂量反应曲线线性最佳,相关系数大于0.98,经实验内10次及不同试验间以16、84、ng/mL测定3次A群多糖中的含量,变异系数在6.3%～11.5%间,回收率在91.8%～105.9%之间,符合常规质控要求,检测限量为4 ng/mL。采用该法测定3批ACYW135群四价脑膜炎球菌多糖疫苗中A群多糖含量、分子大小及回收率的结果均符合规程草案质量标准。结论建立的双抗体夹心ELISA法可尝试用于ACYW135群脑膜炎球菌多糖疫苗中A群多糖的关键质量指标的检测。     

Nucleotide sequence analysis of the sialyltransferase genes of meningococcal serogroups B,C, Y and W135

A rapid method for serogrouping meningococci is essential for the characterization of phenotypically non-groupable meningococcal isolates and clinical samples, particularly for public health management purposes. The Scottish Meningococcus and Pneumococcus Reference Laboratory (SMPRL) provides serogrouping results of meningococcal isolates and clinical samples using a PCR assay which detects restriction fragment length polymorphisms in meningococcal serogroups B, C, Y and W135. Although this PCR system was invaluable when first introduced, it has several drawbacks and lacks the required sensitivity for detecting DNA in clinical samples. Due to the recent introduction of the meningococcal group C conjugate vaccine and an impending group B vaccine, a more robust and informative method for serogroup determination is required. A protocol was devised allowing PCR amplification of the siaD gene of serogroup B, C, Y and W135 meningococci. This system was multiplexed and allowed serogroup differentiation between serogroups B and C and also between B/C and Y/W135 by product size analysis. A nested stage was incorporated into the system for enhanced detection of meningococci in clinical samples, and finally a sequencing protocol was designed allowing detection of any nucleotide changes within the siaD gene. This system allows rapid serogrouping results for use within an agarose gel system as well as more informative results when used for sequencing within the siaD gene.     

Enzyme-linked immunosorbent assay for determination of cyclosporin a in the whole blood

A test-system based on enzyme-linked immunosorbent assay (ELISA) for quantitative determination of cyclosporin A (CSA) in human whole blood has been developed. The detection limit of the method was 25 ng/ml, the linearity of the method in the concentration range of 60–1400 ng/ml varied from 94 to 105%, the variation coefficient did not exceed 8%. The novel method exhibited good correlation with radioimmunoassay and polarization fluoroimmunoassay methods; the linear regression coefficients were 0.965 and 0.984, respectively. The developed test system is stable for at least 9 months when stored at 4°C and can be used in clinical practice.     

Production of recombinant fragment of VacA protein and development of non-invasive method for the diagnosis of Helicobacter pylori infection

Vacuolizing toxin (VacA) Helicobacter pylori is an important factor of pathogenicity of Helicobacter pylori and a basic marker in the diagnosis of helicobacteriosis and related diseases. A coagulation-based diagnostic test-system was elaborated for the detection of VacA in clinical samples. A fragment of vacA was cloned, for the purpose, in Escherichia coli and expressed in preparative quantities; the coded protein was purified and used in raising the diagnostic serum. The thus designed coagulating test-system was successfully tested under the modeling conditions with clinical samples. Therefore, the designed express method can be used for the invasion-free determination of VacA in patients with gastric and duodenal pathologies.     

Antigenic affinity of meningococci and human A- and B-group erythrocytes

The use of the modified method of isohemagglutinin adsorption by microbial antigens in experiments with the causative agent of meningococcal infection has led, for the first time, to the detection of meningococcal antigens affined to the antigens of human erythrocytes, groups A and B. The antigenic affinity of group A erythrocytes and meningococci has proved to be more pronounced in meningococcal strains isolated from the spinal fluid of patients than in cultures obtained from the nasopharynx of healthy persons. The detection of the affinity of these antigens makes it possible to explain the mechanism of differences in the susceptibility of persons with different blood groups to meningococcal infection by "antigenic mimicry".     

The role and place of serologic research in the system of epidemiologic surveillance of meningococcal infection

The results of serological surveys, carried out with a view to the detection of capsular meningococcal polysaccharides in the passive hemagglutination (PHA) test and covering 1,289 patients with systemic meningococcal infection, 221 patients with bacteriologically confirmed meningococcal nasopharyngitis, 2,820 persons in organized groups of children and adults with different epidemiological situation (including 650 carriers) and 4,050 residents of 8 cities with different morbidity levels, were analyzed. The patients, the carriers and the members of organized groups underwent multiple surveys. As shown in this study, the confirmation of the meningococcal etiology of the systemic forms of meningococcal infection (meningitis, meningococcemia) in all cases with the exception of children under 1 year of age, as well as the determination of the groups of meningococci, could be achieved in the PHA test twice as frequently, i.e. in 60-70% of cases, as with the use of the bacteriological method. The same regularities were observed in nasopharyngitis. The conclusion was made on the possibility of using the PHA test for more exact determination of the group of meningococci, prevailing at the initial stage of diseases, this determination playing an essential role in the epidemiological surveillance. The data obtained as the result of serological surveys in organized groups may lead to conclusions on the circulation of meningococci and their group composition, thus showing the necessity of profound epidemiological study with ensuring epidemic-control measures. The selective serological study of some hundreds of donor sera is not expedient in big cities, as it provides no data on the circulation of meningococci there.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibody studies in mice of outer membrane antigens for use in an improved meningococcal B and C vaccine

Abstract Since 1988, N. meningitidis , B:4:P1.15, ET-5 complex, has been responsible for an epidemic of meningococcal disease in Greater São Paulo, Brazil. Despite current trials to develop an effective vaccine against group B meningococci, children less than 2 years old have not been protected. It has been suggested that iron-regulated proteins (IRPs) should be considered as potential antigens for meningococcal vaccines. The vaccines under study consisted of outer-membrane vesicles depleted of lipooligosaccharide from three serogroup B strains and one serogroup C strain, IRPs, meningococcal group C polysaccharide and aluminum hydroxide. Four different protein and C polysaccharide concentrations were studied. The ELISA and bactericidal results showed a higher antibody response when 2 injections of 2.0 μg doses were administered. Despite higher IgG reactivity against antigen preparations containing IRPs seen in ELISA, the bactericidal activity was not increased if the target strain was grown in iron-restricted medium. The influence of addition of alkaline-detoxified lipooligosaccharide (dLOS) on immunogenicity of the vaccine was also investigated, and the dLOS provided for a more functionally specific antibody response.     

Enzyme indicator of the immunobiologic activity of antigens

The test-system has been elaborated for the determination of the ability of different antigens in the cultures of the mouse peritoneal macrophages. Measuring an intracellular acid phosphatase activity, reflecting the extent of the cell activation, was taken as a principle of this test-system. With the help of this test-system ability of a few antigens (lipopolysaccharide, polysaccharide, glycoprotein, protein and low molecular weight substances--peptide and glycopeptide) have been studied. It is testified that above mentioned antigens possessed different effects on the acid phosphatase activity.     

Epitope specificity of murine and human bactericidal antibodies against PorA P1.7,16 induced with experimental meningococcal group B vaccines

Synthetic peptides derived from the predicted loops 1 and 4 of meningococcal PorA, sero-subtype P1.7,16, were used to study the epitope specificity of murine and human PorA P1.7,16 bactericidal antibodies. The predicted loops 1 and 4 are surface exposed and carry in their apices the sero-subtype epitopes P1.7 (loop 1) or P1.16 (loop 4), respectively. Peptides were synthesized as mono- and multimeric peptides. Murine monoclonal and polyclonal antibodies were induced with meningococcal whole cell preparations. Polyclonal antibodies were evoked in volunteers after one immunization with 50 μg or 100 μg protein of a hexavalent meningococcal PorA vesicle vaccine. The induction of PorA antibodies was determined in ELISA using purified PorA P1.7,16. The epitope specificity of anti-PorA antibodies for both murine and human antibodies could be demonstrated by direct peptide ELISA using overlapping multimeric peptides almost spanning the entire loops 1 or 4 of the protein. The capacity of peptides to inhibit the bactericidal activity of murine and human antibodies was investigated using meningococcal strain H44/76 (B:15:P1.7,16) as a target strain. Bactericidal activities could be inhibited with both monomeric and multimeric peptides derived from epitopes P1.7 and P1.16.     

Microchip analytic system for multiplex analysis by real-time polymerase chain reaction with reagents immobilized in microreactors

A microchip analytic system that uses a silicon chip with immobilized in microreactor test-system for multiplex analysis of DNA by real-time polymerase chain reaction (real time PCR) was developed and optimized. We suggested the method of immobilization of PCR-components of a test-system, chose the stabilizer, and conducted the optimization of the composition of reaction mixture to achieve permanent stability of a microchip. We conducted optimization of preparation of samples using magnetic sorbent and indicated that, with 2.6 × 10⁴ copies/ml, 60 min are necessary to obtain positive identification including time for preparation of model samples. The abilities of the created system were demonstrated on the example of microchip analysis of samples with different content of DNA, low absolute limits of detection (20 DNA copies in microreactor), and high reproducibility of the analysis.     

Biosensor analysis of the interaction of potential dimerization inhibitors with HIV-1 protease

Inhibitors of protein-protein interactions are currently considered as perspective prototypes of a new generation of drugs. The most attractive targets for such inhibitors are the oligomeric enzymes which active sites are formed by amino acid residues from different subunits. HIV-1 protease (HIVp), which is active only as a homodimer form, is the classic example of such enzymes. We have developed a new approach for experimental screening of HIVp dimerization inhibitors. It is based on an original biosensor test-system for differential analysis of interaction of tested substances with HIVp dimers and monomers. Using this test-system we have analyzed the most perspective candidate substances predicted by the method of virtual screening, and also some derivatives of glycyrrhizin, triterpenic and steroid glycosides. In the results of this study we have found one compound, which preferentially interacts with HIVp monomers and inhibits in vitro activity of this enzyme with the IC₅₀ value of about 10^?6 M.