Development of a dot enzyme immunoassay for dengue 3: a sensitive method for the detection of antidengue antibodies

Partially purified DEN3 virus was used as antigen in a sensitive dot enzyme immunoassay (DEIA) for the detection of antibodies to flavivirus antigens. We describe here the method used to prepare and optimise the antigen-bearing nitrocellulose membranes and present the results obtained from screening 20 acute phase sera from patients shown to have had recent dengue infections by the haemagglutination inhibition (HI) test. Sixteen pairs of acute and convalescent sera from dengue-negative patients had no detectable antibody to dengue virus by HI. These were shown to have no antibody detectable by DEIA. Sera positive for dengue antibodies by HI had DEIA titers ranging from 10 to several thousand times greater than the titers detected by HI.     

Hemadsorption immunosorbent technique for the detection of dengue immunoglobulin M antibody.

We developed a highly specific, sensitive, and economical hemadsorption immunosorbent technique for the detection of dengue-specific immunoglobulin M (IgM) antibody. The technique is based on the reaction of human sera with anti-human IgM immobilized onto a solid phase followed by the detection of dengue-specific IgM by the addition of a known quantity of dengue virus hemagglutinin and goose erythrocytes. Dengue-specific IgM-positive sera showed hemadsorption. IgM antibody specific for dengue virus was detected in 22 of 39 (56%) convalescent-phase sera from primary dengue infections and 8 of 10 (80%) convalescent-phase sera from secondary dengue infections. Additionally, 32 of 76 single sera from patients were positive for dengue IgM; these sera were previously uninterpretable by the hemagglutination inhibition test, as only a single serum specimen was available. No false-positive results were obtained with sera that were negative by the hemagglutination inhibition test for dengue virus. Crude dengue virus hemagglutinin preparations could be used without purification. Dengue-specific IgG did not interfere with the results, nor was there any cross-reactivity between dengue hemagglutinins and IgM specific for other viruses. Some cross-reactivity of the dengue-specific IgM was observed with Japanese encephalitis virus hemagglutinins, but this did not present any problems in the interpretation of results. This test is specific, inexpensive, highly reproducible, and simple to perform.     

Antibody-capture ELISA for detection of immunoglobulin M antibodies in sera from Japanese encephalitis and dengue hemorrhagic fever patients

Immunoglobulin M (IgM) antibody titers in paired sera from 19 encephalitis and 44 dengue hemorrhagic fever (DHF) patients in Thailand and 42 Japanese encephalitis (JE) patients in Japan were measured by the antibody capture ELISA and applied to distinguish JE virus infection from dengue virus infection. Titer distribution and the ratio of the titers against JE and dengue antigens led to the following diagnostic criteria. The specimens can be considered as positive with JE when IgM-ELISA titer showed over 200 against JE and 4-fold or more higher than titers against any types of dengue antigens. The specimens can be considered as positive with dengue infection when IgM ELISA titer showed over 200 against one of the 4 types of dengue antigens and 4-fold or more higher than against JE antigen. Based on these criteria, 41 of 42 patients in Japan and 11 of 19 encephalitis patients in Thailand could be diagnosed as having JE virus infection while 2 of 19 encephalitis patients in Thailand and 26 of 44 DHF patients in Thailand could be diagnosed as having dengue virus infections.     

Autoimmunity in Aleutian disease: contribution of antiviral and anti-DNA antibody to hypergammaglobulinemia.

The contributions to Aleutian disease gammopathy of specific antiviral antibody and an autoimmune component, anti-DNA antibody, were studied with pastel ranch mink naturally infected with Aleutian disease virus. Specific antibody activities were determined by countercurrent immunoelectrophoresis and radioimmune assay, respectively. Gamma globulin levels (percent gamma) were determined by serum electrophoresis. Within an infected mink population, it was possible to predict the level of gammopathy from measurement of the two antibody levels. For the mink serum samples used, there was better correlation between anti-DNA antibody levels and total serum immunoglobulin than between anti-Aleutian disease virus antibody titers and percent gamma. With serum samples taken over a 2-week interval, significant increases were measured in anti-DNA antibody and percent gamma. Increases in anti-Aleutian disease virus titers during this period were not significant. The results suggest that the continuing increases in serum immunoglobulin in Aleutian disease virus-infected mink are due to both a specific antiviral response and an autoimmune response, as reflected in generation of anti-DNA antibody.     

The defined antigen substrate sphere system with direct immunohistoperoxidase for detection of soluble dengue antigen in sera of patients with dengue hemorrhagic fever.

The defined antigen substrate sphere system is a simple method for detecting antigen or antibody in the circulation. The technic is based on the coupling of antigen or antibody with Sepharose 4B beads that have been activated by cyanogen bromide. In this study the activated beads were exposed to dengue antigen in the serum from a patient with dengue hemorrhagic fever and then stained with antidengue antibody conjugated with horseradish peroxidase. The positive reaction showed brown beads by light microscopy, whereas the negative reaction gave colorless beads. The authors examined 134 specimens from 91 cases. The results were positive in 53.85%. The dengue antigen appeared in the sera on the day before shock or subsidence of fever. The percentages of sera containing soluble dengue antigen were greatest on the day of shock or subsidence of fever (33.33%) and on the fifth day of fever (28.07%). The highest titers of soluble dengue antigen (1:40 to 1:80) appeared in the sera of patients who had Grade III disease on the day of shock. The dengue antigen appeared most often in sera that had high titers of dengue antibody. It is postulated that this detected dengue antigen may be a part of soluble immune complexes formed during the hyperimmune stage of the immune response, and plays a significant role in the pathogenesis of dengue hemorrhagic fever and shock syndrome.     

Quantitative immunoelectrophoretic analysis of human antibodies against herpes simplex virus antigens.

By use of crossed immunoelectrophoresis with intermediate gel, antidbody titers against six individual herpes simplex virus (HSV) glycoproteins and two nonglycosylated proteins were determined in 100 human sera. High antibody titers were found against two different HSV type-common glycoproteins designated Ag8 and Ag11 (containing glycosylated polypeptides D and B, respectively). The anti-Ag8 and -Ag11 titers correlated with HSV neutralizing antibody titers. Most of the serological cross-reactivity between HSV type 1 and type 2 was probably caused by antibodies to Ag8 and Ag11. Human antibodies against one HSV type 1-specific glycoprotein (Ag6, containing glycosylated polypeptide C) and two HSV type 2 glycoproteins (Ag4 and Ag9) were also demonstrated, and the titers correlated better with neutralizing antibody titers of the homologous than of the heterologous virus type. The data presented can be directly applied to the further development of diagnostic reagents.     

Evaluation of an IgG enzyme-linked immunosorbent assay for dengue diagnosis.

BACKGROUND: The hemagglutination inhibition (HI) test has been one of the standards, with the IgM antibody capture ELISA (MAC-ELISA), for the diagnosis of dengue virus infections. The spread of dengue throughout the world and the increasing number of cases to be tested makes an ELISA-format test for IgG antibodies to replace the HI test highly desirable. OBJECTIVES: Evaluate the use of the IgG-ELISA as a substitute for the HI test in dengue diagnosis. STUDY DESIGN: Paired serum samples defined as being from primary or secondary dengue virus infections by HI, were tested by an ELISA that detects IgG antibodies. The correlations of titers and serologic interpretations between these two tests were examined. RESULTS: The IgG-ELISA showed a low correlation with the HI in primary infections, and a higher correlation in secondary infections because of the influence of IgM antibodies in the HI test. Nevertheless, IgG ELISA titers could be reliably associated with primary or secondary infections when analyzed by days after onset of symptoms, and can be used to characterize the immune response after flavivirus infections. CONCLUSION: The combination of the IgM and IgG ELISAs may be used to serologically diagnose dengue virus infections, since the IgG ELISA can substitute for the HI test in characterizing the immune response to dengue virus infections.     

Neutralizing antibody responses to varicella-zoster virus.

Neutralization of varicella-zoster (V-Z) virus by human sera and immune rhesus monkey sera was enhanced by fresh guinea pig complement. There was no marked difference in the degree to which complement enhanced neutralization by sera from current V-Z virus infections and sera from long-past varicella infections. Immunoglobulin G neutralizing antibody in sera from varicella cases was enhanced by complement to a slightly higher degree than was immunoglobulin M (IgM) antibody, and immunoglobulin G neutralizing antibody in immune monkey sera was enhanced to a much greater degree than was IgM antibody. There was a rapid decline in the complement requirement of IgM neutralizing antibodies over the course of immunization of the rhesus monkeys. V-Z neutralizing antibody titers in the presence of complement were higher than complement-fixing titers of the same sera in all groups of individuals studied. IgM neutralizing antibody for V-Z virus was demonstrable in all cases of varicella but in only 1 of 22 zoster cases, and V-Z IgM neutralizing antibody was not detectable in primary herpes simplex virus infections in which heterotypic antibody titer rises occurred to V-Z virus. Complement-fixing antibody for V-Z virus was absent in 19S serum fractions which contained IgM neutralizing antibody for the virus.     

MAC-ELISA and ELISA inhibition methods for detection of antibodies after yellow fever vaccination

The IgM antibody capture ELISA (MAC-ELISA) and ELISA inhibition methods for the detection of antibodies against dengue virus were modified to detect antibodies against yellow fever virus. Tests were carried out in 21 persons vaccinated with 17D and compared with the Plaque reduction neutralizing test. Of 17 naive subjects vaccinated, 16 (94%) seroconverted using the MAC-ELISA test and 14 (82%) seroconverted (or ≥fourfold titer increase) in the ELISA inhibition method. Cross-reactivity was evaluated by both tests and resulted in a high specificity to IgM antibodies against yellow fever, when all the samples from vaccinated individuals were negative by MAC-ELISA using dengue antigen. However, 10.7% of the positive dengue sera from the Santiago de Cuba epidemic cross-reacted by MAC-ELISA using yellow fever antigen. ELISA inhibition method showed high cross-reactivity when the 21 sera pairs were worked with yellow fever and dengue antigens. The MAC-ELISA and ELISA inhibition methods have become indispensable tools in our laboratory in order to maintain a surveillance system for dengue and dengue hemorrhagic fever. They are relatively rapid, simple, and they do not require sophisticated equipment. Both MAC-ELISA and ELISA inhibition methods for yellow fever could be useful for diagnosis, surveillance and yellow fever vaccine evaluation.     

Identification of measles virus-specific hemolysis-inihibiting antibodies separate from hemagglutination-inhibiting antibodies.

The occurrence of antibodies giving hemolysis inhibition (HLI) but not hemagglutination inhibition (HI) was examined in human convalescent and rabbit hyperimmune sera. HI antibodies, which through their interaction with hemagglutinin components display HLI activity, were removed by absorption with Tween 80-ether (TE)-treated measles virus material. This absorption did not change the titer of non-HI HLI antibodies. After removal of HI antibodies from 16 late measles convalescent sera and three batches of gamma globulin. HLI antibody titers showed a two- to eightfold reduction. The titers of neutralizing antibodies were reduced from 1/4 to 1/20 of the original titers. There was a good correlation between the titers of neutralizing and HLI antibodies both in sera from which HI antibodies had been removed by absorption and in sera spontaneously showing markedly higher HLI than HI antibody titers. HLI antibodies with these characteristics could be identified in HI tests when whole virus instead of TE-treated material was used an antigen and anti-antiserum was added to the tests. In contrast to the situation in human sera, antibodies remaining after removal of HI antibodies from rabbit hyperimmune sera against purified virus particles were detectable in neutralization and HLI tests only in the presence of anti-antiserum. However, virus particles from which the major fraction of all envelope projections had been removed by treatment with 0.004% trypsin induced the production of non-HI HLI antibodies active also in the absence of anti-antiserum. TE and formalin treatment destroyed the hemolytic activity of virus preparations and also their capacity to induce a production of non-HI HLI antibodies.     

Metabolic inhibition test for louping-ill,measles and herpes simplex virus in plastic panels

Summary A metabolic inhibition test in plastic panels with HeLa cells and a continuous line of human amnion cells was developed for titration of louping-ill, herpes simplex and measles viruses and antibodies. The loupingill and herpes simplex virus titers were the same or slightly lower, and measles virus titers considerably lower in panel titration than in tube titration based on the cytopathogenic effect. In the neutralization tests with various human sera and animal immune sera, the louping-ill neutralization titers were about the same in panel and tube titrations. The herpes simplex and measles panel neutralization titers were comparable with the corresponding complement-fixing antibody titers.In HeLa cells with lactalbumin hydrolysate medium herpes simplex virus caused a clear metabolic inhibition, but in Eagle's minimum essential medium the diluted virus increased cell metabolism.This study was supported by grants from the Rockefeller Foundation and the Sigrid Juselius Foundation.     

Cross-reactivity in flavivirus serology: new implications of an old finding?

To investigate the influence of pre-existing antibodies against tick-borne encephalitis (TBE) or yellow fever (YF) viruses on dengue virus antibody test results, we examined sera from vaccinees and from individuals with previous TBE virus infection. Distinct IgG antibody cross-reactivity was found in about 15.1% in the YF-vaccinated group and in about 9.5% in the TBE-vaccinated group. Altogether 15 out of a total of 80 samples tested (18.8%) had detectable dengue virus IgG antibody titres. The serum samples from patients with acute TBE virus infection not only had the highest anti-TBE virus antibodies but were also highly cross-reactive against dengue virus antigens. The high cross-reactivity rate of YF and TBE antibody-positive sera in dengue virus antibody assays should be taken into account in the interpretation of laboratory tests for the diagnosis of flavivirus infections and when undertaking seroepidemiological surveys.     

Demonstration by immunoelectro-osmophoresis of precipitating antibodies to a purified rubella virus antigen.

The nonionic detergent Triton X-100 was used to extract antigens of rubella virus from infected tissue culture cells. Three virus-specific antigens were demonstrated by crossed immunoelectrophoresis by using a pool of human gamma globulin as antiserum. The most dominant of these antigens were purified by ion-exchange chromatography on diethylaminoethyl-cellulose. This antigen was of glucoprotein nature and had slow electrophoretic motility and low binding capacity to diethylaminoethyl-cellulose. Thus, it seems likely that the antigen is identical with the precipitating antigen of rubella virus designated b-antigen or tro-osmophoresis with precipiting antibody in sera obtained from patients recovering from acute postnatal rubella. The precipitin reaction that could be correlated to the hemaglutination-inhibition titers of the same sera appeared 12 days after onset of the disease and remained positive for several years.     

Isolation and characterization of a relevant Aspergillus fumigatus antigen with IgG- and IgE-binding activity

An immunologically relevant antigen fraction was isolated from a cytoplasmic extract of Aspergillus fumigatus mycelium. The fraction was obtained by concanavalin-A affinity chromatography and hydrophobic interaction chromatography. Two protein bands were discernible in polyacrylamide gel electrophoresis while two precipitin peaks were detected in crossed immunoelectrophoresis. When tested against sera from patients with Aspergillus-induced diseases, the fraction showed both IgG and IgE antibody-binding activity. All of the sera studied from patients with allergic bronchopulmonary aspergillosis (ABPA) showed high IgG and IgE antibody titers, while sera from patients with aspergilloma and cystic fibrosis with ABPA demonstrated high IgG titers and only a moderate increase in IgE titers. None of the other groups showed any significant antibody titers against this antigen in their sera. Because of its binding to both IgG and IgE antibodies this fraction was found to be useful in the immunodiagnosis of Aspergillus-induced diseases.     

重组登革病毒E蛋白结构域Ⅲ抑制登革病毒感染的初步研究

目的 了解原核表达的登革病毒(dengue virus, DV)的E蛋白结构域Ⅲ直接抑制登革病毒感染及其抗体的中和作用.方法 在大肠埃希菌中表达1~4型登革病毒E蛋白结构域Ⅲ(EⅢ).重组蛋白纯化后,进行阻断DV-2感染BHK~21细胞试验.用重组蛋白制备免疫血清,检测抗体中和作用.结果 在大肠埃希菌中成功表达了1-4型登革病毒E蛋白结构域埃希菌,4型重组E蛋白结构域Ⅲ均能够阻断2型DV感染,4型重组蛋白的免疫血清均能中和2型DV,但中和抗体效价不同.结论 原核表达的登革病毒结构域Ⅲ可以直接抑制病毒感染,所产生的抗体具有中和作用.直接抑制和中和抗体均对同型病毒作用较强.     

Evaluation of immunoglobulin M and G capture enzyme-linked immunosorbent assay Panbio kits for diagnostic dengue infections.

BACKGROUND: Serological assays are widely used to confirm dengue virus infections and to differentiate between a primary and a secondary infection. OBJECTIVE: Two commercial dengue diagnostic kits, Panbio Dengue IgM Capture and Dengue IgG Capture ELISA (Brisbane, Australia) were evaluated. STUDY DESIGN: Three hundred and seventy-three serum samples were tested. Panel sera included samples from dengue confirmed cases (representing both primary and secondary infections), from non-dengue infectious diseases, and from healthy individuals. The MAC-ELISA/Dengue IPK was used for the detection of anti-dengue virus IgM antibody in the sera and the ELISA inhibition method (EIM/Dengue IPK) was used to differentiate between primary and secondary infections. Both these reference assays, which were previously developed in the Arbovirus Laboratory at the "Pedro Kouri" Tropical Medicine Institute, were employed as the gold standard. RESULTS: High sensitivity (96.8%) and specificity (99.4%) were found with the commercial diagnostics when compared to the reference methods. Furthermore, high concordance 95.5% in classifying dengue infection types (primary or secondary infections) was observed. CONCLUSIONS: The Panbio Dengue IgM and IgG assays offer a good alternative for dengue diagnosis. They are easy to perform and results can be obtained in less than 3h.     

Human antibody to influenza C virus: its age-related distribution and distinction from receptor analogs.

Sera from persons of four age groups (1 to 2 years, 2 to 5 years, 20 to 30 years, and 65 to 85 years) were analyzed for hemagglutination inhibition (HI) activity for influenza C virus. Significant HI activity was found in 66% of the 237 sera tested, and titers ranged from 8 to 512. In the yoiung adult group, 96% had antibody and the highest mean titer (74.7) of any age group. Positive sera were far less common in young children (36 to 47%), and relatively low titers (18.3) were common among adults over 65. The high percentage of sera with antibody to influenza C virus suggests that infections with this virus occur at a rate greater than previously recognized. The high percentage of young adults with elevated levels of HI antibody suggested either that an immune response to influenza C infections is common or that the observed HI activity might be attributable, in part at least, to nonspecific inhibitors in the sera. We showed both directly and indirectly that most if not all the inhibitory activity in the human sera we examined was due to specific antibody, mostly immunoglobulin G. This conclusion is based on the finding that the single serum protein fraction with HI activity was found to have a molecular weight equivalent to that of 7S antibody (150,000) and that the HI activity was removed by absorption to staphyloccal protein A. Moreover, immunoglobulin from only HI-positive sera bound specifically to cells infected with influenza C virus, as shown by inhibition of hemadsorption and immunofluorescence. These findings were supported by similar results obtained with chicken antisera to C virus.     

Serological evidence of dengue fever among refugees, Hargeysa, Somalia

Epidemics of a malaria-like illness affected several thousand residents of the Dam Camp, a refugee camp near Hargeysa in Somalia, during 1985, 1986, and 1987. The disease was characterized by fever, chills, sweats, headache, back and joint pains for as long as 10 days in some patients. Blood smears from acutely ill patients were negative for malaria. Of 28 acute and 10 convalescent sera tested by the indirect fluorescent antibody (IFA) and by the hemagglutination inhibition (HI) tests, all were negative for antibody to Rift Valley fever, Crimean-Congo hemorrhagic fever, Sindbis, Chikungunya, yellow fever, and Zika viruses. However, antibody reactive to dengue 2 virus was detected by the IFA test in 39% (15/38), and 11 of 29 (38%) of the same sera were antibody positive by the HI test. Also, IgG antibody reactive to dengue 2 was demonstrated in 60% (17/28) of the same sera by the enzyme immunoassay (EIA), and 14% (4/28) were positive for IgM antibody. Of ten patients for which acute and convalescent sera were available, two developed four fold or greater rises in antibody titer evidencing infection. These data suggested that dengue virus may have been the cause of the epidemic among the Dam Camp refugees.     

Antibody response to the rubella virus structural proteins in infants with the congenital rubella syndrome

Forty-five serum samples from 31 newborns and infants with the congenital rubella syndrome (CRS) were tested by immunoprecipitation to determine their antibody spectra to each of the structural proteins of rubella virus. Most sera (37/45) contained little or no E2 protein-specific antibody, but some (6/45) precipitated a greater amount of the E2 glycoprotein than the E1 glycoprotein. The relative E1/E2 ratio was found to decrease with time when serial serum samples from the same patient were tested. No correlation between the IgG class hemagglutination inhibition antibody titers and the E1/E2 ratio could be demonstrated. However, in some serum specimens relatively high neutralizing antibody titers were correlated with immunoprecipitation of the E2 glycopolypeptide. None of the CRS sera reacted well with the C protein. The immunoprecipitation patterns found in CRS sera were qualitatively different from those observed in a series of 25 sera from young adults with conventional serologic evidence of rubella immunity following natural infection. All of the natural immune sera recognized each of the three structural polypeptides of rubella virus.     

深圳地区人和鸡群中H9N2亚型流感病毒病原学和血清流行病学调查

目的：了解甲型流感病毒N9N2亚型毒株在深圳地区鸡群和人群中的分布。方法：采用常规的鸡胚双腔法来分离病毒。抗体测定，采用红细胞凝集抑制（HI）试验和中和试验测定法。结果：从深圳地区农贸市场鸡群中分离到27株H9N2亚型流感病毒，但未能从人群中分离到H9N2病毒。约有26％人血清中检测到H9亚型毒株的抗体，（HI滴度≥20），同时还发现抗体阳性率和几何均数随人群年龄增长而增高，同时与职业有关。然而，在鸡群中H9毒株的抗体阳性率仅为7％。结论：禽H9N2毒株不仅能感染人，而且在深圳地区人群和禽类中较为广泛的分布。人H9N2很大可能来源于鸡的H9N2毒株。