RESUMEN

Primary human cytomegalovirus (HCMV) infection during pregnancy is a frequent cause of fatal damage in populations with low prevalence of HCMV. Differentiation of primary vs. recurrent HCMV infection is an important issue in prenatal counseling. Antibodies specific for viral glycoproteins become detectable only with considerable delay with relation to HCMV infection or IgG seroconversion. Thus, lack of glycoprotein specific (gp-specific) antibodies can serve as a convenient indicator to identify those pregnant women that bear an elevated risk for HCMV transplacental transmission and fetal sequelae. In the opposite case, presence of gp-specific antibodies virtually excludes HCMV primary infection several weeks before sampling. However, no standardized screening assay for HCMV gp-specific antibodies had been available thus far. For this reason, an ELISA based on procaryotically expressed fragments of HCMV glycoprotein B (gB; gpUL55) was developed. Small fragments of gB from two different laboratory strains, encompassing the antigenic domain 2 (AD2) sufficed for sensitive and specific detection of gp-specific antibodies. The gB-ELISA titers correlated with titers of virus neutralizing antibodies in serum samples from primary or recurrent HCMV infections. Seroconversion kinetics of the gB-ELISA in samples from patients with primary HCMV infection closely paralleled the delay in seroconversion of gp-specific antibodies as determined by neutralization assay. Thus this assay provides a diagnostic tool that is easy to perform and can significantly add to available methods for the timely identification of primary HCMV infection during pregnancy. In addition, the gB-ELISA may be helpful in other clinical settings for the differentiation of primary HCMV infection from diseases caused by other pathogens.

Asunto(s)

Antígenos Virales/inmunología , Infecciones por Citomegalovirus/diagnóstico , Citomegalovirus/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas del Envoltorio Viral/inmunología , Enfermedad Aguda , Anticuerpos Antivirales/sangre , Citomegalovirus/genética , Infecciones por Citomegalovirus/sangre , Diagnóstico Diferencial , Humanos , Pruebas de Neutralización , Recurrencia

RESUMEN

Serological tests based on the antibodies directed against the Epstein-Barr virus early antigen (EA) and viral capsid antigen (VCA), which have been recognized as tumor markers for nasopharyngeal carcinoma (NPC), are routinely used to help in the diagnosis of this malignancy. The detection of these antibodies reveals very low titers, found only in a small proportion of young compared with older NPC patients. This is a problem for the diagnosis of NPC, especially among Maghrebians, among whom young people are also affected, and emphasizes the necessity to search for more reliable markers. The present study reports results of immunoglobulin G (IgG) and IgA responses of NPC patients to recombinant EA antigens p54 (BMRF1) and p138 (BALF2), VCA complex antigens p18 (BFRF3) and p23 (BLRF2), and EBNA antigen p72 (BKRF1). Our results show that IgA-EA-p54 and -p138 (IgA-EA-p54+138) antibodies have a diagnostic value for detection of NPC (70%), compared with IgA-VCA-p18+23 and IgA-EBNA-p72, which have limited diagnostic value, especially in young patients. It is also noteworthy that IgA-EA-p54+138 can detect a high percentage (64%) of NPC cases negative by immunofluorescence. These results, however, clearly show that a single test cannot achieve the objective of detecting all NPC patients, and it seems advisable to combine different tests for the diagnosis of NPC. The combination of IgG-ZEBRA with IgA-EA-p54+138 improved the sensitivity of detection of NPC to 95% in the overall NPC population. The use of IgA-EA-p54+138 in combination with IgG-ZEBRA will facilitate detailed studies on the pattern of antibody response, which may result in the development of useful serological markers to guide the treatment of NPC.

Asunto(s)

Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Herpesvirus Humano 4/inmunología , Neoplasias Nasofaríngeas/diagnóstico , Adolescente , Adulto , Antígenos Virales/genética , Niño , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Persona de Mediana Edad , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , Transactivadores/genética , Transactivadores/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología

RESUMEN

Timely and reliable detection of acute primary human cytomegalovirus (HCMV) infection is important in prenatal screening programs and for differential diagnosis of infectious mononucleosis-like disease. Enzyme-linked immunosorbent assays (ELISAs) based on HCMV proteins enable the sensitive detection of immunoglobulin M (IgM) antibodies during primary infection. However, concerns have been raised about possible cross-reactivities of the HCMV antigens used for the design of such ELISAs with IgM antibodies induced by Epstein-Barr Virus (EBV). In this study we investigated whether IgM antibodies generated during acute EBV infection reacted with recombinant HCMV antigens. Serum samples from patients with primary EBV infection frequently scored positive when tested in different HCMV IgM ELISAs, irrespective of whether conventional or recombinant antigens were used for the design of the HCMV IgM assays. Such cross-reactive IgM antibodies were found to be directed against short glycine-rich motifs contained within the nonstructural HCMV proteins pUL44 and pUL57. Further analyses revealed that these glycine-rich motifs were major antigenic domains for IgM antibodies induced during HCMV infection. Their deletion from recombinant proteins abrogated reactivity with IgM synthesized during HCMV infection. EBV-induced IgM antibodies that reacted with HCMV antigens showed similar kinetics of reactivity in HCMV- or EBV-specific assays in the course of primary EBV infection, indicating that the two populations of antibodies were highly overlapping. The results demonstrate that primary EBV infection leads to the induction of IgM antibodies that specifically bind to widely used diagnostic antigens of HCMV. This has to be considered in the interpretation of HCMV-specific IgM assays.

Asunto(s)

Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos/inmunología , Antígenos Virales/inmunología , Citomegalovirus/inmunología , Proteínas de Unión al ADN/inmunología , Herpesvirus Humano 4/inmunología , Proteínas Virales/inmunología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Antígenos Virales/genética , Reacciones Cruzadas , Proteínas de Unión al ADN/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Glicina/inmunología , Inmunoglobulina M/inmunología , Cinética , Datos de Secuencia Molecular , Polímeros , Proteínas Virales/genética

RESUMEN

A new pair of Epstein-Barr virus ELISAs (Biotest Anti-EBV VCA IgG and VCA IgM ELISA) was evaluated for usefulness for routine diagnosis of acute EBV infections. The ELISAs are based on two viral capsid antigens (VCA), p23 (BLRF2, full-length) and p18 (BFRF3, carboxy-half), that are combined by autologous gene fusion. In total, 179 sera were tested in direct comparison with classical VCA immunofluorescence assays (IFA). With the help of clinical data and additional reference serology, i.e., heterophile antibodies, anti-EA IgG (IFA) and anti-EBNA-1 IgG (ELISA), the patients were divided into the following categories: seronegatives (46), acute primary infections (67), previous infections (39), suspected reactivations (20) and constellations with intermediate serological patterns (7). The VCA IgG and VCA IgM ELISAs showed overall agreement to IFA of 95.0% and 94.4%, respectively. The calculated analytical performance (sensitivity; specificity) of VCA IgG and VCA IgM was 94.0%; 97.8% and 97.1%; 96.5%, respectively. A certain delay in seroconversion of anti-p23-p18 IgG may account for a significant difference in sensitivity of the VCA IgG ELISA between primary (88.4%) and previous infections (100%). In summary, the new recombinant VCA ELISAs yielded good correlation to VCA IFA and in combination with EBNA-1 IgG allow rapid, sensitive, and specific diagnosis of infectious mononucleosis or EBV immune status in general.

Asunto(s)

Anticuerpos Antivirales/sangre , Cápside/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Infecciones por Virus de Epstein-Barr/diagnóstico , Herpesvirus Humano 4/inmunología , Adolescente , Adulto , Antígenos Virales/inmunología , Niño , Preescolar , Infecciones por Virus de Epstein-Barr/sangre , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Fragmentos de Péptidos/inmunología , Proteínas Recombinantes de Fusión/inmunología , Sensibilidad y Especificidad , Pruebas Serológicas

RESUMEN

Human Herpesvirus 8 (HHV-8) is clearly associated with Kaposi's sarcoma (KS), body cavity-based lymphomas (BCBL), and certain forms of multifocal Castleman's disease (MCD). It appears to be the sexually transmissible agent involved in the development of AIDS-associated KS. HHV-8 genomes are invariably present in BCBL-derived cell lines where lytic replication of the virus can be induced by phorbol esters (PE). First-generation HHV-8 serological assays were based on these cell lines. More recently, several genes encoding HHV-8 antigens have been identified. One of the most reactive antigens is encoded by HHV-8 open reading frame K8.1. Although K8.1 does not exhibit overt sequence homology to any other known gene, it is likely to be analogous to gp220/350 of Epstein-Barr or gp150 of murine herpesvirus-68, virion-envelope glycoproteins involved in target cell recognition. Mice were immunized with purified GST-K8.1 fusion protein expressed in E. coli. After fusion of murine plasma cells with the myeloma cell line P3-X63-Ag8. monoclonal antibodies (MAbs) were generated, which are specifically directed against K8.1 protein. The binding site for each MAb was identified by deletion mutant analysis using recombinant GST-K8.1 mutants and K8.1-specific peptides. Without exception, the epitopes recognized by these MAbs were located within the N-terminal part of the protein [amino acids (aa) 29 to 80], thus identifying a highly immunogenic region. These antibodies will not only be useful tools for HHV-8 diagnostics, but will also facilitate the analysis of K8.1 function.

Asunto(s)

Anticuerpos Monoclonales/biosíntesis , Epítopos/inmunología , Glicoproteínas/inmunología , Herpesvirus Humano 8/inmunología , Proteínas del Envoltorio Viral/inmunología , Proteínas Virales , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Sitios de Unión de Anticuerpos/inmunología , Western Blotting , Cartilla de ADN/química , Ensayo de Inmunoadsorción Enzimática , Epítopos/genética , Femenino , Técnica del Anticuerpo Fluorescente , Expresión Génica , Vectores Genéticos , Glutatión Transferasa/metabolismo , Glicoproteínas/genética , Herpesvirus Humano 8/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas del Envoltorio Viral/genética

RESUMEN

Several diagnostic tools are available for the identification of acute and latent viral infections. Although newly developed nucleic acid amplification methods, such as the polymerase chain reaction (PCR), have proved to be very useful diagnostic procedures, conventional methods, such as cell culture and serology, still play an important role in viral diagnostics. Despite the fact that modern serological assays, such as enzyme-linked immunosorbent assay (ELISA), are inexpensive and easy to perform, there is a strong demand to improve the performance of such systems. Most serological tests are based on poorly characterized antigens produced in infected culture cells. It has been shown, however, that only few viral antigens contained in these preparations are essential for serodiagnosis. In addition, numerous viral proteins display homologies with their counterparts from related viruses. Finally, the specificity of serological assays can also be reduced by contaminating proteins from host cells. Selective purification of natural viral antigens using, for example, immunoaffinity chromatography is one possible way to improve the quality of an antibody assay. However, the low concentration of most viral proteins in cell culture-derived antigen preparations reduces the practicability of this approach.

RESUMEN

Using recombinant 15- to 30-kDa fragments and fusion with glutathione S-transferase (GST), we investigated the seroreactivity of three large structural proteins of Epstein-Barr virus (EBV), p150 (BcLF1, capsid), p143 (BNRF1, tegument), and gp125 (BALF4, membrane) in Western blots. None of 13 fragments tested, however, was qualified for diagnostic application. In contrast, the two small viral capsid antigens (VCA), p18 (BFRF3) and p23 (BLRF2), demonstrated sensitive (100%) EBV-specific immunoglobulin G (IgG) reactivities. While p18 additionally showed maximum sensitivity for IgM detection, the IgM sensitivity of p23 was restricted (44%). An autologous fusion protein, p23-p18, which consists N-terminally of full-length p23, followed by the carboxy half of p18, was constructed. This antigen was subjected to indirect VCA enzyme-linked immunosorbent assays (ELISAs), for IgG and IgM, as well as to a micro-capture (microc) IgM ELISA. All assays were found to be 100% specific when EBV-negative sera were tested. Using sera from previously infected individuals, the p23-p18 fusion revealed an improved IgG sensitivity of 99% compared to sensitivities of 97 and 93% for the single antigens p18 and p23, respectively. The sensitivity and specificity of the indirect IgM ELISA with samples of primary and past infections, respectively, were 100%. The microc principle for IgM overcame completely the interference by rheumatoid factors. Compared to the specificity of the indirect IgM version, the specificity with sera collected from rheumatoid arthritis patients increased from 48 to 100%. In summary, the p23-p18 IgG and microc IgM ELISAs showed excellent performances and are promising new diagnostic tests for the detection of EBV-specific antiviral capsid antibodies.

Asunto(s)

Anticuerpos Antivirales/sangre , Cápside/inmunología , Infecciones por Herpesviridae/diagnóstico , Herpesvirus Humano 4/inmunología , Fragmentos de Péptidos/inmunología , Proteínas Recombinantes de Fusión/inmunología , Infecciones Tumorales por Virus/diagnóstico , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Pruebas Serológicas

RESUMEN

The open reading frames ORF 52, ORF 65, K12, and K8.1 of the human herpesvirus 8 (HHV8) were expressed as glutathione-S-transferase (GST) fusion proteins and analysed by Western blotting (WB) and enzyme-linked immunosorbent assay (ELISA). The open reading frame (ORF) 65 and K8.1 antigens gave the highest reactivity (71%) in sera from HIV-dependent Kaposi's sarcoma (KS) patients. Therefore both antigens appear to be essential for HHV8 diagnostics, whereas ORF K12 and ORF 52 were of minor importance. Using polymerase chain reaction (PCR) out of the peripheral blood of these KS patients, 48% were detected as positive. By testing an N-terminal-deleted construct (amino acid 80-171) of ORF 65, we could show that the N-terminal region of this protein is essential to mediate full immunogenic reactivity. By analysing different deletion mutants of ORF K8.1, the major epitope was found to be located between aa 29 and 101. The prevalence of antibodies directed against the different antigens was determined for healthy blood donors to be 3-6%. The different antibody patterns obtained in HIV-patients with and without KS support the hypothesis that different antibody profiles develop during the course of KS.

Asunto(s)

Antígenos Virales/inmunología , Herpesvirus Humano 8/inmunología , Inmunoglobulina G/inmunología , Southern Blotting , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Glutatión Transferasa/genética , Infecciones por VIH/virología , Humanos , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/inmunología

RESUMEN

Portions of three human cytomegalovirus (HCMV) polypeptides, which were shown previously to be highly reactive with patient sera, were expressed in Escherichia coli as autologous fusion proteins. Purified recombinant polypeptides were used as antigens in enzyme linked immunosorbent assay (ELISA) and compared against assays which use natural viral antigen from cell culture for their ability to improve IgM-specific serology of acute HCMV-infection. A fusion protein (CM2) which contained two copies of the C-terminal portion of pUL44 (p52, aa 297-433) and one copy of a highly reactive fragment of the major DNA-binding protein pUL57 (aa 545-601) proved to be superior in sensitivity and specificity compared to assays which use culture derived antigen. A construct expressing one copy of the fragments from pUL44 and pUL57 in fusion with the 54 amino terminal residues of pUL32 (pp150, aa 994-1048) did not lead to an improved sensitivity compared to CM2. Adversely, this polypeptide reacted with a number of sera from asymptomatic blood donors infected latently with HCMV indicating low specificity of this antigen for the detection of acute infection. Concordant results were obtained with an antigen that combined only the C-terminal portions of pUL44 and pUL32 (CM3). ELISA experiments with sequential sera from renal transplant recipients demonstrated that detection of IgM-antibodies using CM2 as antigen correlated closely with acute infection, whereas high levels of IgM-antibodies against CM1 and CM3 persisted for a month following acute HCMV-infection. These results indicate that the application of a single autologous fusion protein like CM2 as antigen for recombinant ELISAs can improve significantly IgM-serodiagnosis of acute HCMV infection.

Asunto(s)

Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Infecciones por Citomegalovirus/virología , Citomegalovirus/aislamiento & purificación , Proteínas de Unión al ADN/inmunología , Epítopos Inmunodominantes/inmunología , Inmunoglobulina M/inmunología , Fosfoproteínas , Proteínas de la Matriz Viral/inmunología , Proteínas Virales/inmunología , Anticuerpos Antivirales/sangre , Clonación Molecular , Citomegalovirus/inmunología , Infecciones por Citomegalovirus/sangre , Infecciones por Citomegalovirus/diagnóstico , Infecciones por Citomegalovirus/inmunología , Proteínas de Unión al ADN/genética , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Humanos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Pruebas Serológicas , Proteínas de la Matriz Viral/genética , Proteínas Virales/genética

RESUMEN

The immunoglobulin A-specific reactivities of recombinant viral proteins from nine different reading frames of human cytomegalovirus were evaluated in enzyme-linked immunosorbent assay experiments. Antigen fragments of reading frames pUL32, pUL44, and pUL57 were identified as preferable antigens for immunoglobulin A serodiagnosis. Application of autologous fusion proteins which combine these polypeptides may be useful especially for the early detection of acute secondary human cytomegalovirus infection.

Asunto(s)

Anticuerpos Antivirales/sangre , Antígenos Virales , Infecciones por Citomegalovirus/diagnóstico , Citomegalovirus/inmunología , Inmunoglobulina A/sangre , Pruebas Serológicas/métodos , Enfermedad Aguda , Antígenos Virales/genética , Citomegalovirus/genética , Infecciones por Citomegalovirus/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Estudios de Evaluación como Asunto , Humanos , Sistemas de Lectura , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Factores de Tiempo

RESUMEN

Reactivation of the Epstein-Barr virus was reported to occur frequently under immunosuppressive therapy following organ transplantation. However, little is known about the clinical significance of these EBV reactivations. Therefore, we searched for correlations among the treatment with various immunosuppressive drugs, the incidence of CMV infections, rejection crises, and serological signs of EBV reactivation. EBV-specific antibodies were measured with novel ELISAs, utilizing the recombinant antigens p72 (for anti-EBV nuclear antigen [EBNA]1-IgG), p54, and p138 (anti-early antigen [EA]-IgM, -IgG, -IgA) in a follow-up study of 79 renal transplant recipients. Patients receiving antithymocyte globulin or antilymphocyte globulin therapy showed increasing anti-EA-IgG and -IgA more often than did patients not receiving antithymocyte globulin or antilymphocyte globulin therapy (P < 0.05). In patients receiving OKT3 antirejection therapy, anti-EA-IgM seroconversion was found more frequently (P < 0.01). A significant correlation was also found between groups of patients who had had at least one rejection episode versus patients without any sign of organ rejection, and the incidence of increasing anti-EA-IgG (P < 0.05). Since in most of these patients signs of EBV reactivation followed the appearance of the rejection episode, this may not be due to viral-induced rejection but may be caused by the reinforced immunosuppression during antirejection therapy. As opposed to patients with no signs of CMV infection and with nonsymptomatic CMV infection, patients undergoing symptomatic CMV infection showed anti-EA-IgM seroconversion (P < 0.01), increasing anti-EA-IgA (P < 0.01), and decreasing anti-EBNA-IgG (P < 0.01) more frequently. Our results confirm the role of immunosuppressive therapy in the pathogenesis of EBV reactivation. We further demonstrate a striking coincidence of EBV reactivation and symptomatic CMV infection.

Asunto(s)

Infecciones por Citomegalovirus/etiología , Rechazo de Injerto , Herpesvirus Humano 4/fisiología , Trasplante de Riñón/efectos adversos , Activación Viral , Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Humanos , Inmunoglobulina M/sangre , Inmunosupresores/farmacología

RESUMEN

A small polypeptide from pUL57 of human cytomegalovirus was identified as a major target for the immunoglobulin M antibody response. This antigen seems to be superior to antigenic fragments from pp150 and p52 in the identification of sera from acutely infected patients. It may therefore represent an essential antigen for recombinant immunoglobulin M antibody tests for human cytomegalovirus.

Asunto(s)

Antígenos Virales , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Proteínas de Unión al ADN/inmunología , Proteínas Virales/inmunología , Enfermedad Aguda , Anticuerpos Antivirales/sangre , Infecciones por Citomegalovirus/diagnóstico , Infecciones por Citomegalovirus/microbiología , Humanos , Inmunoglobulina M/sangre , Proteínas Recombinantes de Fusión/inmunología

RESUMEN

DNA fragments from eight different reading frames of human cytomegalovirus (HCMV) were generated by PCR and subsequently cloned and expressed in Escherichia coli in fusion with glutathione S-transferase. The recombinant viral antigens were evaluated in immunoblot analyses. The most reactive antigens were purified and further evaluated in ELISAs. For this, sera from healthy blood donors and immunocompetent individuals with acute HCMV infection, and follow-up sera from transplant recipients with acute primary HCMV infection were used. The results of our experiments indicate that only three particular recombinant polypeptides from two viral proteins are necessary for serodiagnosis. While a fragment covering amino acids (aa) 495 to 691 of pp150 (150/1) was the most suitable antigen for the identification of infected individuals in general, immunoglobulin M antibodies against the C-terminal parts of pp150 (aa 862 to 1048; 150/7) and p52 (aa 297 to 433; 52/3) proved to be excellent serological markers to monitor acute HCMV infection. The selected recombinant antigens enable the improvement of serodiagnosis of HCMV-related diseases, especially during the early stages of infection.

Asunto(s)

Infecciones por Citomegalovirus/diagnóstico , Ensayo de Inmunoadsorción Enzimática/métodos , Pruebas Serológicas/métodos , Anticuerpos Antivirales/sangre , Antígenos Virales/genética , Clonación Molecular , Citomegalovirus/genética , Citomegalovirus/inmunología , Infecciones por Citomegalovirus/etiología , Infecciones por Citomegalovirus/inmunología , ADN Recombinante/genética , ADN Viral/genética , Escherichia coli/genética , Estudios de Evaluación como Asunto , Glutatión Transferasa/genética , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Trasplante de Riñón/efectos adversos , Sistemas de Lectura , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología

RESUMEN

A population of 2,024 voluntary blood donors was tested for anti-EBNA-1 IgG and anti-VCA IgG serum antibodies to define the EBV infection rate and to compare two different epidemiological markers. Initial screening was performed with a sensitive EBNA-1-IgG ELISA based on recombinant antigen (Biotest) and a VCA-IgG ELISA based on conventional antigen. Both ELISAs had concordant results in 90.4% of the sera. The infection rate was found to be 96.8%. The expected immune status VCA+/EBNA+ was observed in 98.1% of the seropositives. The comparison of sensitivity, specificity, and predictive values between the two screening assays underlines the superiority of the EBNA-1 ELISA. The marker anti-EBNA-1 IgG as detected by a sensitive ELISA (Biotest anti-EBV recombinant) is suitable for defining previous EBV infection (positive predictive value 99.8%). The high infection rate in the adult population, however, renders the supply with EBV-negative blood rather difficult.

Asunto(s)

Anticuerpos Antivirales/sangre , Donantes de Sangre , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Infecciones por Herpesviridae/diagnóstico , Infecciones por Herpesviridae/epidemiología , Herpesvirus Humano 4 , Inmunoglobulina G/sangre , Adulto , Ensayo de Inmunoadsorción Enzimática/métodos , Reacciones Falso Positivas , Humanos , Selección de Paciente , Sensibilidad y Especificidad

RESUMEN

A new Epstein-Barr virus (EBV) ELISA system (Biotest Anti-EBV recombinant) was evaluated for usefulness for routine diagnosis of EBV primary infection. The assay system is composed of three different microtest plates coated with three highly purified recombinant EBV antigens. The early antigens p138 (BALF2, truncated) and p54 (BMRF1, whole sequence) are used as a mixture for testing IgM (assay 1) and IgG (assay 2) antibodies. In addition, the EBNA-1 antigen p72 (BKRF1, carboxy-half) is used for detecting IgG antibodies (assay 3). Three panels of sera were examined in direct comparison with standard immunofluorescence (IF): Specimens of (i) 120 infectious mononucleosis (IM) patients, (ii) 60 patients with acute CMV infection, toxoplasmosis or rheumatic disease, respectively, and (iii) 185 healthy blood donors as a control group. 119 IM patients were clearly recognized as having acute primary infection (sensitivity 99.2% compared to VCA-IgM by IF). Three apparently false-positive results were obtained with patients of other diseases and none within the control group (specificity 98.8%). The data suggest that the recombinant ELISA can be used advantageously for standardized rapid diagnosis of acute EBV primary infection.

Asunto(s)

Anticuerpos Antivirales/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Herpesvirus Humano 4/inmunología , Mononucleosis Infecciosa/diagnóstico , Adolescente , Adulto , Antígenos Virales/inmunología , Niño , Preescolar , Estudios de Evaluación como Asunto , Humanos , Mononucleosis Infecciosa/inmunología , Juego de Reactivos para Diagnóstico , Proteínas Recombinantes/inmunología

RESUMEN

DNA-fragments coding for a variety of different viral antigens have been cloned and expressed in E. coli. Selected purified recombinant antigens were used for detection of specific antibodies by the means of ELISA technique. This approach has been used for the development of four different ELISA's for the detection of HIV- and EBV-specific antibodies.

Asunto(s)

Anticuerpos Antivirales/análisis , Antígenos Virales , Ensayo de Inmunoadsorción Enzimática , Virosis/diagnóstico , Especificidad de Anticuerpos/inmunología , Antígenos Virales/inmunología , Anticuerpos Anti-VIH/análisis , Antígenos VIH , Infecciones por VIH/diagnóstico , Infecciones por VIH/inmunología , VIH-1/inmunología , Herpesvirus Humano 4/inmunología , Humanos , Mononucleosis Infecciosa/diagnóstico , Mononucleosis Infecciosa/inmunología , Proteínas Recombinantes , Infecciones Tumorales por Virus/diagnóstico , Infecciones Tumorales por Virus/inmunología , Virosis/inmunología

RESUMEN

DNA fragments coding for a variety of different viral antigens have been cloned and expressed in Escherichia coli. Selected purified recombinant antigens were used for detection of specific antibodies by means of the ELISA technique. This approach has been used for the development of four different ELISAs for the detection of HIV- and EBV-specific antibodies.

Asunto(s)

Anticuerpos Antivirales/análisis , Antígenos Virales , Ensayo de Inmunoadsorción Enzimática , Anticuerpos Anti-VIH/análisis , Herpesvirus Humano 4/inmunología , Proteínas Recombinantes , Western Blotting , Epítopos/inmunología , VIH-1/inmunología , VIH-2/inmunología , Humanos

RESUMEN

Four recombinant, diagnostically useful Epstein-Barr virus (EBV) proteins representative of the viral capsid antigen (p150), diffuse early antigen (p54), the major DNA-binding protein (p138), and the EBV nuclear antigen (p72) (W. Hinderer, H. Nebel-Schickel, H.H. Sonneborn, M. Motz, R. Kühbeck, and H. Wolf, J. Exp. Clin. Cancer Res. 7[Suppl.]:132, 1988) were used to set up individual enzyme-linked immunosorbent assays (ELISAs) for the qualitative and quantitative detection of immunoglobulin M (IgM) and IgG antibodies. In direct comparison with results obtained by standard immunofluorescence or immunoperoxidase assays, it was then shown that the recombinant EBV ELISAs provide the means for specific and sensitive serodiagnosis of infectious mononucleosis (IM) caused by EBV. The most useful markers in sera from such patients proved to be IgM antibodies against p54, p138, and p150. Additional positive markers for recent or ongoing IM apparently were IgG antibodies against p54 and p138. In contrast, anti-p72 IgG had a high preference for sera from healthy blood donors and, therefore, can be considered indicative of past exposure to the virus. Altogether, the individual ELISAs proved to be as specific and at least as sensitive for the diagnosis of IM as the currently available standard techniques are. Moreover, our findings suggest that, by combining individual test antigens, a workable ELISA system consisting of three assays (IgM against p54, p138, and p150; IgG against p54 and p138; and IgG against p72) can be established for the standardized rapid diagnosis of acute EBV infections.

Asunto(s)

Mononucleosis Infecciosa/diagnóstico , Adolescente , Adulto , Anticuerpos Antivirales/sangre , Antígenos Virales , Biomarcadores/sangre , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática , Estudios de Evaluación como Asunto , Femenino , Herpesvirus Humano 4/inmunología , Humanos , Inmunoglobulina G/análisis , Inmunoglobulina M/análisis , Mononucleosis Infecciosa/inmunología , Masculino , Pruebas Serológicas , Proteínas Virales/inmunología

RESUMEN

The Epstein-Barr virus nuclear antigen I (EBNA I) is the only latent EBV antigen consistently expressed in malignant tissues of the nasopharynx. A 20-amino-acid synthetic peptide, p107 contains a major epitope of EBNA I. We tested sera from 210 patients with nasopharyngeal carcinoma (NPC) and from 128 normal individuals (NHS) for IgA antibodies to p107 using an enzyme-linked immunosorbent assay (ELISA). Whereas 191/210 (91%) of NPC patients had IgA antibodies to p107, only 17/128 (13.3%) of NHS had such antibodies and only 6/57 (10.5%) of sera from patients with malignancies other than NPC had IgA-p107 reactivity. Thirty-nine salivary samples from 46 NPC patients (84.8%) also contained IgA-p107 antibodies whereas only 3/42 (7.1%) of normal saliva samples were IgA-p107 positive. The results suggest that IgA antibodies to EBNA I may become a useful, easily measurable, marker for NPC.

Asunto(s)

Anticuerpos Antineoplásicos/análisis , Anticuerpos Antivirales/análisis , Antígenos Virales/inmunología , Carcinoma/inmunología , Epítopos/inmunología , Herpesvirus Humano 4/inmunología , Inmunoglobulina A/análisis , Neoplasias Nasofaríngeas/inmunología , Saliva/inmunología , Adulto , Ensayo de Inmunoadsorción Enzimática/métodos , Antígenos Nucleares del Virus de Epstein-Barr , Humanos , Péptidos/inmunología , Proteínas Recombinantes/inmunología , Valores de Referencia , Proteínas Virales/inmunología

RESUMEN

Cell-suspension cultures of Ascochyta rabiei-resistant (ILC 3279) and -susceptible (ILC 1929) chickpea (Cicer arietinum L.) cultivars were compared with regard to their elicitor-induced accumulation of pterocarpan phytoalexins and increases in the activities of biosynthetic enzymes. The growth performances and protein patterns of the two cell-culture lines were essentially identical. Treatment of cell cultures with a polysaccharide elicitor from A. rabiei induced fivefold-higher amounts of the phytoalexins medicarpin and maackiain in the cells of the resistant than in the susceptible cultivar. Glucose 6-phosphate dehydrogenase and eight enzymes representing the general phenylpropanoid pathway, the flavonoid-forming steps and the pterocarpanspecific branch of phytoalexin biosynthesis were found to be elicitor-induced. Phenylalanine ammonia-lyase and chalcone synthase reached sharp, transient optima some 8 h after elicitor application in the cells of both cultivars. The activities of isoflavone 2'- and 3'-hydroxylases were only induced in cells of the resistant cultivar with a maximum after 8 h. Cinnamic acid 4-hydroxylase, chalcone isomerase, 2'-hydroxyisoflavone reductase and pterocarpan synthase showed a later or no sharp optimum. The isoflavone-specific 7-O-glucosyltransferase was not induced in either cell-culture line. Cells of the susceptible cultivar failed to induce significant activities of isoflavone 2'-hydroxylase and these cells produced only very low amounts of phytoalexins. Isoflavone 2'-hydroxylase is postulated to be the main limiting enzyme for pterocarpan biosynthesis in cells of the susceptible cultivar. The pterocarpan biosynthetic pathway in chickpea cells represents a suitable model for investigations of differential gene activation in connection with the expression of antimicrobial defence reactions.