Maan Zrein

Evaluation of INNO-LIA Syphilis Assay as a Confirmatory Test for Syphilis

ABSTRACT We evaluated the sensitivity and specificity of a new confirmatory test for treponemal antibodies, INNO-LIA Syphilis (Innogenetics NV, Ghent, Belgium), on a large number of sera from a clinical laboratory. This multiparameter line immunoassay (LIA) uses recombinant and synthetic polypeptide antigens derived from Treponema pallidum proteins. In a single-blinded cross-sectional retrospective study, 289 seronegative sera, 219 seropositive sera, and 23 sera with an indeterminate serological status for syphilis were analyzed. All sera were tested by the T. pallidum hemagglutination assay (TPHA), the immunoglobulin (IgG)-fluorescent T. pallidum absorption assay (IgG-FTA-ABS), and the Venereal Disease Research Laboratory (VDRL) test. In addition, some seropositive samples were analyzed by the 19S-IgM-FTA-ABS test, an enzyme immunoassay (IgM-EIA), and the MarDx immunoblotting assay. Based on a consensus diagnosis derived from conventional serology, all of the sera were classified as positive, negative, or indeterminate, and the results were compared with the findings of the INNO-LIA Syphilis assay. The sensitivity and specificity of the LIA were 100% (219 of 219) and 99.3% (286 of 288), respectively. Compared to TPHA and IgG-FTA-ABS, the new test gave a significantly higher number (P = 0.021 and P < 0.0001, respectively) of correct results than either of the other two tests. The multiparameter INNO-LIA Syphilis assay is a useful confirmatory test for syphilis because it increases the reliability of syphilis diagnosis with respect to current conventional techniques.

Identification of immunoreactive regions of rubella virus E1 and E2 envelope proteins by using synthetic peptides

Relatively large (16-33 aa) synthetic peptides (SPs) representing defined sequences of rubella virus (RV) E1 and E2 envelope proteins were used in lymphocyte stimulation and enzyme immunoassays to map immunoreactive regions recognized by peripheral blood mononuclear cells (PBMNC) and serum antibodies from healthy RV-seropositive, RV-seronegative, and RV-vaccinated adults. Five distinct immunoreactive regions were identified in RV E1 protein, spanning residues (11-39), (154-179), (199-239), (226-277), and (389-412), which stimulated cellular responses in 29-83% of the subjects tested. Two SPs, E1(213-239) and E1(258-277) containing previously-identified virus neutralizing antibody domains, reacted with serum antibodies and also stimulated lymphoproliferation suggesting that these E1 sequences contain linked or overlapping B-and T-cell antigenic sites. The frequency and magnitude of cellular responses to E2 SPs were somewhat lower. SPs encompassing E2 residues (50-72), (140-199), and (244-263) stimulated lymphocyte responses in 28-64% of the subjects tested, while to a lesser degree, SPs within residues (1-36) were also stimulatory. E2 SPs within the regions (1-36), (151-170), and (244-263) also showed low levels of antibody reactivity with sera from RV-seropositive subjects. E2(244-263) which induced the highest level of response among the E2 SPs tested, was of interest due to previous reports of sequence homology of this RV region with human myelin and its potential immunopathogenic role in demyelinating autoimmune diseases. Identification of these potentially immunodominant regions of RV envelope proteins is an important first step in the rational design of new RV vaccines.

Analysis of overlapping T- and B-Cell antigenic sites on rubella virus E1 envelope protein influence of HLA-DR4 polymorphism on T-cell clonal recognition

Abstract A CTL antigenic site located between residues 273 and 291 of the E1 envelope protein of RV was identified by 51Cr-release assays employing SPs. Two E1-specific CTL clones were examined for immune recognition of RV wild-type and attenuated vaccine strains and recombinant E1 protein. The exact sequence (273–284) recognized by both clones was delineated by using truncated and overlapping SPs covering these residues. The defined T-cell site overlapped almost completely with a virus neutralizing antibody-binding site previously identified with mouse monoclonal and human antibodies. A series of single aa-substituted SP analogues of E1(273–284) was used to define residues critical for T-cell recognition. Using EBV-Bl displaying different HLA-DR haplotypes and -DR4 subtypes as targets to determine MHC class II restriction elements, immune recognition by both T-cell clones was shown to be associated with HLA-DR4. Three HLA-DR4 subtypes (DR4Dw 13A, DR4Dw13B, and DR4KT2) sharing a common residue, glutamic acid at position 74 in their β 1 chains, were able to present SP E1(273–284) to the T-cell clones.

Quantitation of rheumatoid factors by enzyme immunoassay using biotinylated human IgG

An enzyme-linked immunosorbent assay (ELISA) using biotinylated human IgG for the detection of rheumatoid factor (RF) in human sera has been developed. This assay avoids some of the problems encountered with earlier ELISA methods used for RF detection. Accurate RF titers of human sera were determined using a simple computer program developed for curve fitting of ELISA data.

Development of a Novel Multiplex Immunoassay Multi-cruzi for the Serological Confirmation of Chagas Disease.

Background Chagas disease is due to the parasite Trypanosoma cruzi, a protist disseminated by a Triatome vector. This disease is endemic to Latin America and considered by WHO as one of the 17 world’s neglected diseases. In Europe and in North America, imported cases are also detected, due to migration of population outside of the endemic region. Diagnosis of T. cruzi infection is usually made indirectly by the detection of specific antibodies to T. cruzi antigens. Following initial diagnostic evaluation or screening test (qualifying or discarding blood donation), a confirmation test is performed for samples initially reactive. The test presented in this study aims at the confirmation/refutation of the infectious status of human blood samples and will permit taking appropriate clinical measures. Methodology/Principal Findings We designed a novel array of twelve antigens and printed these antigens onto 96-well plates. We tested 248 positive samples T. cruzi, 94 unscreened blood donors’ samples from non-endemic area, 49 seronegative blood donors, 7 false-positive and 3 doubtful samples. The observed reactivities were analyzed to propose a decision-tree algorithm that correctly classifies all the samples, with the potential to discriminate false-positive results and sticky samples. We observed that antibodies levels (Sum of all antigens) was significantly higher for PCR positive than for PCR negative samples in all studied groups with Multi-cruzi. Conclusion/Significance The results described in this study indicate that the Multi-cruzi improves the serological confirmation of Chagas disease. Moreover the “sum of all antigens” detected by Multi-cruzi could reflect parasitemia level in patients–like PCR signals does—and could serve as an indicator of parasite clearance in longitudinal follow-ups. Validation of this assay is still required on an independent large collection of well characterized samples including typical false-reactive samples such as Leishmaniasis.

Detection of Rheumatoid Factors of Different Isotypes by Elisa Using Biotinylated Avian Antibodies

An enzyme-linked immunosorbent assay (ELISA) using biotinylated avian antibodies has been developed for the detection of rheumatoid factors (RF) of various isotypes in human sera. The avian antibodies were obtained from the egg yolks of hens immunized with human IgA, IgG and IgM and were purified by affinity chromatography. These reagents made it possible to detect each of the three isotypes of RF present in the same serum and avoided false positives since the Fc epitopes of avian immunoglobulins are not recognized by the antibody combining site of RF.

Anti-Trypanosoma cruzi cross-reactive antibodies detected at high rate in non-exposed individuals living in non-endemic regions: seroprevalence and association to other viral serologies.

Cross-reactive antibodies are characterized by their recognition of antigens that are different from the trigger immunogen. This happens when the similarity between two different antigenic determinants becomes adequate enough to enable a specific binding with such cross-reactive antibodies. In the present manuscript, we report the presence, at an “abnormal” high frequency, of antibodies in blood samples from French human subjects cross-reacting with a synthetic-peptide antigen derived from a Trypanosoma cruzi (T. cruzi) protein sequence. As the vector of T. cruzi is virtually confined to South America, the parasite is unlikely to be the trigger immunogen of the cross-reactive antibodies detected in France. At present, the cross-reactive antibodies are measured by using an in-house ELISA method that employs the T. cruzi -peptide antigen. However, to underline their cross-reactive characteristics, we called these antibodies “Trypanosoma cruzi Cross Reactive Antibodies” or TcCRA. To validate their cross-reactive nature, these antibodies were affinity-purified from plasma of healthy blood donor and were then shown to specifically react with the T. cruzi parasite by immunofluorescence. Seroprevalence of TcCRA was estimated at 45% in serum samples of French blood donors while the same peptide-antigen reacts with about 96% of T. cruzi -infected Brazilian individuals. In addition, we compared the serology of TcCRA to other serologies such as HSV 1/2, EBV, HHV-6, CMV, VZV, adenovirus, parvovirus B19, mumps virus, rubella virus, respiratory syncytial virus, measles and enterovirus. No association was identified to any of the tested viruses. Furthermore, we tested sera from different age groups for TcCRA and found a progressive acquisition starting from early childhood. Our findings show a large seroprevalence of cross-reactive antibodies to a well-defined T. cruzi antigen and suggest they are induced by a widely spread immunogen, acquired from childhood. The etiology of TcCRA and their clinical relevance still need to be investigated.

Use of egg-yolk antibody for detection of respiratory syncytial virus in nasal secretions by ELISA

SummaryEgg-yolk immunoglobulins extracted from the eggs of hens immunized with respiratory syncytial virus (RSV) have been used as a reagent in double sandwich ELISA for detecting RSV in nasal secretions. The sensitivity of virus detection was the same in indirect ELISA, using rabbit anti chicken globulin conjugate, as when biotinylated yolk globulin and labeled avidin were used for detection. The specificity of ELISA for detecting RSV using yolk antibody was similar to that achieved by indirect immunofluorescence using commercial reagents of mammalian origin. Purified immunoglobulin was easily extracted from egg yolk; the amount of globulin present in a single preparation obtained from a batch of ten eggs was sufficient to carry out 106 ELISA tests for RSV detection.

Use of synthetic peptides to map regions of rubella virus capsid protein recognized by human T lymphocytes

Synthetic peptides (SPs), 18-29 amino acids long, representing selected sequences of rubella virus (RV) capsid (C) protein were used in lymphocyte proliferation assays to identify antigenic regions recognized by T lymphocytes from healthy RV-reactive adults. Four SPs, C(1-29), C(90-114), C(108-134) and C(255-300), stimulated proliferation of peripheral blood mononuclear cells and RV-specific T-cell lines from the same donors. C(1-29V), an SP analogue containing an RA27/3 RV vaccine strain sequence, stimulated higher levels of proliferation in T cells obtained from RV-vaccinated subjects than did the comparable wild-type (M33 strain) RV sequence.

Cellular hyperimmunoreactivity to rubella virus synthetic peptides in chronic rubella associated arthritis.

OBJECTIVES--Immune recognition of the major structural proteins of rubella virus by peripheral blood mononuclear cells and synovial inflammatory infiltrates of a patient with documented chronic rubella associated arthritis was compared with responses of normal healthy rubella virus immunoreactive subjects to establish if there were unusual response patterns associated with rubella associated arthritis in this subject. METHODS--Synthetic peptides (16-33 amino acids in length) representing selected amino acid sequences of the rubella virus envelope (E1 and E2) and capsid (C) proteins were used in lymphocyte stimulation assays with peripheral blood mononuclear cells or synovial inflammatory infiltrates to determine T lymphocyte recognition of antigenic sites within the synthetic peptides. A rubella virus specific polymerase chain reaction was used to determine the persistence of rubella virus in the patients cells. RESULTS--The patients peripheral blood mononuclear cells showed abnormally increased lymphoproliferative responses to three E1 synthetic peptides encompassing residues 219-234, 389-411, and 462-481, and one E2 synthetic peptide containing the sequence 50-72, of which the last three were predicted to contain T cell antigenic sites. Although the patients peripheral blood mononuclear cells showed positive proliferative responses to C synthetic peptides, these were not unusual. The number of synthetic peptides within the E1, E2, and C panels recognised by the patients peripheral blood mononuclear cells was greater than was previously observed in normal healthy subjects. The recognition of synthetic peptides by synovial inflammatory infiltrates was similar to peripheral blood mononuclear cells but the responses measured were lower. The polymerase chain reaction was negative for rubella virus detection in peripheral blood mononuclear cells and synovial inflammatory infiltrates. CONCLUSIONS--Abnormally increased T cell recognition of antigenic sites within rubella virus E1 and E2 proteins observed in this patient with rubella associated arthritis suggests chronic antigenaemia due to persistent rubella virus in tissue sites other than peripheral blood mononuclear cells or synovial inflammatory infiltrates.