三种ELISA试剂盒检测猪流行性乙型脑炎病毒血清IgG抗体比较

目的 比较国内常用商品化ELISA试剂盒检测猪流行性乙型脑炎病毒血清IgG抗体的诊断效果。方法 选用3种猪乙脑病毒血清IgG抗体ELISA检测试剂盒(Keqian,Lvshiyuan,Tianchen),以微量中和试验为金标准,对90份猪血清进行检测分析。结果 中和试验的阳性检出率为34.4%(31/90),Keqian,Lvshiyuan和Tianchen阳性检出率分别为50.0%(45/90), 47.8%(43/90)和 38.9%(35/90)。灵敏度最高的是Keqian,为90.32%,但其特异度只有71.19%;Tianchen的灵敏度和特异度分别为83.87%和79.66%;Lvshiyuan的灵敏度和特异度分别仅为41.94%和16.95%。与中和试验比较,Tianchen试剂盒的κ系数最高为0.63,其次是Keqian为0.57,Lvshiyuan仅为0.04。显示3种试剂盒具稳定性。结论 目前国内商品化猪乙脑病毒血清IgG抗体ELISA检测试剂盒诊断结果差异较大,与中和试验相比存在较高的假阴性,质量有待提高。     

Absence of antibodies to the human immunodeficiency virus in sera from Africa prior to 1975.

Three different assays for detection of antibodies to the human immunodeficiency virus (HIV) were conducted on 677 sera obtained from 1964 to 1975 from male and female children and adults in Uganda and other countries in Africa. Several sera were collected from individuals with Kaposi sarcoma. No evidence of antibodies to the virus was noted up to 1975. These results strongly suggest that the emergence of HIV in Africa occurred relatively recently. Further studies are required to determine the geographic origin of the acquired immunodeficiency syndrome virus.     

An enzyme-linked immunosorbent assay to characterize dengue infections where dengue and Japanese encephalitis co-circulate

The diagnostic sensitivity and specificity of detection of anti-dengue IgM by antibody capture enzyme-linked immunosorbent assay (ELISA) was investigated in dengue infections in a variety of clinical settings. Sera from uninfected controls were uniformly negative. Serial specimens from experimental and natural infections showed that viremia and fever terminated as anti-dengue IgM became detectable. Anti-dengue IgM appeared in most cases by the 3rd afebrile day of illness and declined to undetectable levels after 30-60 days. Assay sensitivity was 78% in admission sera (924/1,183; 95% CI = 75-81%) and 97% in paired sera (1,030/1,062; 95% CI = 96-98%) thus exceeding or matching the performance of the hemagglutination-inhibition assay. Measurement of the anti-dengue IgM to anti-Japanese encephalitis IgM ratio correctly identified all sera from 112 patients with strictly defined Japanese encephalitis and 98% (307/312; 95% CI = 96-99%) of sera from patients whose dengue infections were confirmed by virus isolation. Dengue infections could be classified as primary or secondary by determining the ratio of units of dengue IgM to IgG antibody. We propose that measurement of dengue and Japanese encephalitis IgM and IgG antibodies upon admission and discharge from hospital care should replace the hemagglutination inhibition assay as the standard dengue serologic technique in regions where these 2 viruses co-circulate.     

Absence of antibodies to cyclic citrullinated peptide in sera of patients with hepatitis C virus infection and cryoglobulinemia

OBJECTIVE: To determine if antibodies to cyclic citrullinated peptide (anti-CCP) are found in chronic hepatitis C virus (HCV) infection. METHODS: Rheumatoid factor (RF) and anti-CCP were measured in sera from 50 patients with HCV infection but without cryoglobulinemia, sera from 29 patients with mixed cryoglobulinemia (including 13 with rheumatic symptoms and 5 with arthritis), and sera from 20 normal blood donors. Anti-CCP was measured by second-generation enzyme-linked immunosorbent assay (ELISA). RESULTS: No sera with elevated anti-CCP were found in patients with HCV infection without cryoglobulinemia, and in that population, the maximum anti-CCP was 10 units, well below the positive cutoff of 20 units. Positive findings on RF testing >13 IU/ml were present in 22 (44%) of the HCV patients, with RF >50 IU/ml in 8 (16%) and a maximum RF of 526 IU/ml. Of the cryoglobulinemia patients, 22 (76%) had positive results on tests for RF, including 18 (62%) with RF >50 IU/ml and a maximum RF of 5,540 IU/ml. Two (6.9%) of the cryoglobulinemia patients had borderline-positive findings on tests for anti-CCP (25 units and 37 units), which were false-positive results caused by nonspecific binding in the ELISA. No association between the RF and the anti-CCP concentrations was found. CONCLUSION: Whereas RF was frequent in patients with HCV infection with and without cryoglobulinemia, anti-CCP was not observed in patients with uncomplicated HCV infection. Borderline-positive anti-CCP results were observed infrequently in patients with mixed cryoglobulinemia and were caused by nonspecific binding to plastic. Measurement of anti-CCP may help in diagnosing RA in patients with chronic HCV infection.     

Leukocyte antibodies in human sera and in immune rabbit sera

A study of leukocyte antibodies is presented using (1) the sera of rabbits immunized with human leukocytes, and (2) the sera of three patients screened forthe presence of such antibodies from among 36 patients with hematologic disease,31 of whom (including the 3 studied in detail) had received multiple transfusions.The following technics are described and were employed: Leukoagglutination,leukoprecipitation including tube and agar-plate methods, agglutination ofantigen-coated tanned and untanned sheep erythrocytes, the effect of antiseraupon phagocytosis of heat-killed staphylococci by leukocytes, and upon ameboidmotility of leukocytes.

The leukoagglutinin test gives reliable clearcut results providing that appropriate controls are included and certain criteria adhered to, in order to facilitatethe recognition of clumping due to other factors than true antigen-antibodyunion.

No leukoprecipitins were detected in human sera with the technics used inthis study. Immune rabbit sera, on the other hand, gave two reaction-lines inagar media, when set up against leukocyte extract.

Immune rabbit sera reacted strongly with antigen-coated tanned sheep redblood cells. Human sera did not so react. One of the three selected human serareacted with antigen-coated untanned erythrocytes, suggesting the presence of apolysaccharide antigen extractable from human leukocytes and capable of stimulating antibody formation in the human. Immune rabbit sera, and otherhuman sera, did not react in this test.

A suggestive but perhaps not a conclusive effect upon phagocytosis of bacteriaby leukocytes exposed to human leukocyte antibody for 1 hour could be demonstrated.

By means of ameboid motility studies, a cytotoxic effect of the human antiseraupon human leukocytes could be demonstrated after 18 hours of incubation, butnot after 3 hours. This was interpreted as evidence of a delayed reaction.

Certain cardinal points from a clinical and theoretical standpoint with regardto the genesis of leukocyte antibodies in man are briefly reviewed. A possibleanalogy between leukocyte antibody formation and the homograft reaction isdiscussed. It is suggested that the rarity of leukocyte iso-antibody formationfollowing transfusion is related to the fact that the intravenous pathway may bea poor route of immunization for these antigens.

Submitted on April 4, 1957 Accepted on July 1, 1957     

Maternally derived antibodies to Japanese encephalitis virus in cattle

Serum samples were collected from 55 pairs of calves that had not passed the first summer and their dams reared in Kagoshima from 1983 through 1985. They were investigated for the HI antibody to Japanese encephalitis virus (JEV) in calves and for the correlation of antibody levels between calves and their dams. The maternally derived antibody was detected in 35 of 55 calves (63.6%), showing the mean antibody titer of 12.4. A significant negative correlation (p less than 0.01) was noted between the age of calve and titer of passively acquired antibodies. The regression equation suggested that the maternal antibody disappeared from the calf serum at about 3 months of age. On the other hand, 37 of 55 dams (67.3%) were positive for HI antibody to JEV, and the mean titer of the antibody was 12.7. There was a significant correlation (p less than 0.01) in serum antibody titers between dams and their calves.     

Seroepidemiological survey on dengue and Japanese encephalitis virus infections in Asian monkeys

To investigate the ecology of dengue and Japanese encephalitis (JE) viruses in the forest in Asia, a seroepidemiological survey was carried out on 358 Southeast Asian cynomolgus (Macaca iris), 33 Indian bonnet (Macaca radiata) and 37 Japanese (Macaca fuscata) monkey sera by a plaque reduction neutralization test. The results indicated that Southeast Asian monkeys were naturally infected with these viruses but the frequency of antibody to them varied considerably according to the geographical origin of the monkeys. The frequency of antibody to one or more types of dengue virus were 87.2, 49.5, 34.3, 34.2 and 14.9% in Malaysian, Vietnamese, Cambodian, Indonesian and Filipino cynomolgus monkey sera, respectively. None of the Indian bonnet monkey sera neutralized type 1 dengue virus which was the only virus type examined with this monkey species. Monkey sera collected in Japan where dengue virus infection had not been known since 1944 did not significantly neutralize dengue viruses. JE virus antibody was detected at 29.7, 9.0, 8.6, 2.7, 1.4 and 0% in Japanese, Cambodian, Vietnamese, Indonesian, Filipino and Malaysian monkey sera respectively.     

Comparative susceptibility of mosquito species and strains to oral and parenteral infection with dengue and Japanese encephalitis viruses

Thirty-four strains of Asian and Pacific mosquitoes belonging to 22 species of 7 genera were compared for oral and/or parenteral susceptibility to infection with 1 or more strains of each of the 4 dengue serotypes. Surprisingly, several species of common man-biting Aedes were much more susceptible to oral infection with each of the 4 dengue serotypes than was Aedes aegypti. These species included Aedes albopictus and members of the scutellaris group of the subgenus Stegomyia found on South Pacific islands. Mosquito strains and species relatively susceptible to 1 dengue serotype usually were relatively susceptible to the others also. Almost all species of Aedes tested were uniformly susceptible to parenteral infection with the dengue viruses but, with the exception of a species of Tripteroides, species of all other genera were comparatively resistant to that mode of infection. Dengue viruses usually replicated to about the same extent in orally-infected mosquitoes as they did in parenterally-infected specimens of the same species. Seventeen species of mosquitoes of 7 genera also were tested for parenteral susceptibility to infection with Japanese encephalitis virus. With the possible exception of 2 species of Anopheles, the virus replicated to about the same degree in all species tested and achieved levels considerably higher than did any of the dengue viruses in the same mosquito strain and species held under the same conditions.     

The complexity of antibody-dependent enhancement of dengue virus infection

Antibody-dependent enhancement (ADE) has been proposed as a mechanism to explain dengue hemorrhagic fever (DHF) in the course of a secondary dengue infection. Very recently, Dejnirattisai et al., 2010 [1], published an important article supporting the involvement of anti-prM antibodies in the ADE phenomenon. The complexity of ADE in the context of a secondary dengue infection is discussed here.     

Short report: absence of protective neutralizng antibodies to West Nile virus in subjects following vaccination with Japanese encephalitis or dengue vaccines

Protection of individuals against West Nile (WN) encephalitis is an emerging concern in the United States and Europe. We investigated whether immunization with licensed inactivated Japanese encephalitis (JE) vaccine or experimental live attenuated dengue vaccines resulted in induction of cross-neutralizing antibodies against WN virus. Protective neutralizing antibody titers to WN virus were not detected in any volunteer despite successful immunization to related flaviviruses. Vaccination against JE or dengue is unlikely to prevent WN virus infection but may still protect against disease.     

Distribution of dengue and Japanese encephalitis among children in rural and suburban Thai villages

In the rainy season of 1989, IgG and IgM antibodies against dengue and Japanese encephalitis viruses (measured by enzyme-linked immunoassay [ELISA]) in serum from all primary-school children in two areas of central Thailand were sampled in order to choose a study site for more detailed epidemiological and entomological analysis. Students in three schools in the largely non-agricultural, suburban community of Bang Bua Thong, Nontaburi Province were sampled in late June and July. Of 1,477 children, 33/1,000 had recent dengue infection and 7/1,000 had recent JE infection. The rate of dengue infection in each village influenced the rate in schools, in that the rate of the school could be predicted from the proportion of students coming from each village. This result suggested that most transmission occurred in the residential environment; otherwise, the rate in each village going to a single school would be identical. Serum samples were taken in late August in the agricultural community of Hua Samrong, Chachoengsao Province. Of 748 students in two schools, 95/1,000 had signs of recent dengue infection and 32/1,000 had signs of recent JE infection. Two of 12 villages had significantly less flavivirus infection than some other villages and three villages had significantly more flavivirus infection. The children from one village had a dengue infection rate of 256 per 1,000, which was higher than the national average for the worst year (1987) previously recorded in Thailand. Within Hua Samrong, there was evidence for significant dengue transmission in one of the schools and concentrated transmission in small areas of two of the villages. The younger age group (3-8 years old) had significantly higher risk of infection by either flavivirus than older children. Elevated homes with wooden floors had significantly higher risk of dengue in the largest village. The observations from 1989 describe the epidemiological situation in rapidly developing, rural villages. This stage of development is probably being repeated throughout Southeast Asia as formerly isolated, rural villages become connected by transportation and economy to urban centers. What appears to be a single dengue outbreak based on passive surveillance conducted on a regional basis may actually be a variety of epidemiological situations. The practical implication of this conclusion is that application of a combination of vaccination and vector control should be targeted to higher risk areas in order to increase the likelihood of regional dengue virus eradication.     

Monoclonal antibodies for dengue virus prM glycoprotein protect mice against lethal dengue infection

Five murine monoclonal antibodies (Mabs) reactive against the prM glycoproteins of DEN-3 and -4 were used to passively protect mice in vivo against lethal challenge with homologous and heterologous dengue virus serotypes. Four of the 5 prM-reactive monoclonals cross-protected mice against heterologous challenge, whereas 1 protected against challenge with only the homologous serotype. Although in vitro binding to virions was readily demonstrated, only 2 of the prM Mabs had detectable neutralizing activity. The neutralizing activity could not be enhanced by anti-mouse immunoglobulin or complement. However, 4 of the 5 prM Mabs fixed complement. This is the first report of prM-specific Mabs that are protective in mice.     

Immunological analysis of Japanese encephalitis virus using anti-Kamiyama monoclonal antibodies

Fifteen hybridomas were produced by fusing P3X63Ag8.653 mouse myeloma cells with spleen cells from BALB/c mice immunized with Japanese encephalitis (JE) virus Kamiyama strain. Antigenic analysis of twenty-five strains of JE virus was carried out by hemagglutination inhibition (HI) test with anti-Kamiyama monoclonal antibodies (KAMIMAs). Twenty-one JE virus strains were isolated from various parts of Japan, and four foreign countries. These strains had been isolated from different host between 1935 and 1979. According to the HI test against the five species-specific monoclonal antibodies, the twenty-five JE virus strains were classified serologically as follows: Group A: Kamiyama, Sekiya, Mochizuki, Nishizono, Sasazaki, Mie 44-1, Fukuoka 7101, Fukuoka 7202, Fukuoka 7309, Fukuoka 7311, Fukuoka 7452, Fukuka 7463, Fukuoka 7506, Kumamoto 80679, Chang Mai and JaGAr 02 strains. Group B: Nakayama-RFVL and Nakayama-Yoken strains. Group C: Nakayama-Yakken, Kalinina, G-1 late, JaGAr 01, Beijing 1 and 691004 strains. Group D: Muar strain. These results mostly corresponded with the serological classification by anti-Nakayama-RFVL monoclonal antibodies. Analysis of the biological activities of KAMIMAs revealed that there were no correlations among HI titer, ELISA titer and neutralization titer. Neutralizing and some non-neutralizing monoclonal antibodies protected mice infected with lethal doses of JE virus Kamiyama strain. SDS-PAGE and immunoblotting analysis revealed that three antibodies reacted with a 52.0 kD band under non-reducing conditions and with a 53.0 kD band under reducing conditions, five antibodies reacted with a 52.0 kD band only under non-reducing conditions, and that seven antibodies reacted with a 14.5 kD band under both non-reducing and reducing conditions.     

The effect of dengue virus infection on the clinical sequelae of Japanese encephalitis: a one year follow-up study in Thailand.

In order to determine if prior dengue virus infection reduces the severity of Japanese encephalitis (JE), we examined 127 patients hospitalized during the 1970 JE epidemic in the Chiangmai and Lampang Valleys of northern Thailand. Patients were studied during the first 30 days after onset of JE; 120 of these patients were examined one year later for residual neurologic sequelae. About 21% of patients had serological evidence of a prior dengue virus infection. Morbidity and mortality in patients with and without prior dengue virus experience were compared. These comparisons were made within two age groups to exclude differences due to age alone;     

Precipitating antibodies to non-treated dengue type 2 viral antigens in sera of Thai hemorrhagic fever patients by crossed immunoelectrophoresis

Crossed immunoelectrophoresis of dengue type 2 virus revealed at least two precipitating antigens which shared some antigenic determinants. Glycoprotein components of both antigens were detected by binding to concanavalin A. Sera from dengue hemorrhagic fever patients showed precipitating antibodies to both antigens which could be quantitated according to the precipitate patterns formed in the intermediate gel of crossed immunoelectrophoresis. All secondary dengue hemorrhagic fever patients demonstrated an increase in precipitin titers in convalescence sera. Most patients with mild illness contained precipitating antibodies in acute phase sera whereas severe cases did not. Convalescent sera from severe cases showed only low titers. These precipitating antibodies may be associated with protection since they were produced early only in those with mild form of illness.     

Detection of IgM antibodies from cerebrospinal fluid and sera of dengue fever patients.

During the dengue epidemic from late 1987 to 1989, 6 specimens of cerebrospinal fluid (CSF) and sera for IgM detection were collected from 4 cases virologically confirmed dengue patients who had neural symptoms. Another 20 serum specimens, which had been diagnosed as dengue infection either virologically or serologically, were sent to the laboratory from Kaohsiung Medical College Hospital. All these specimens were also taken to detect the existence of IgM. The results showed that IgM could be detected from 14 out of 20 serum specimens. One of the positive specimens showed IgM can last up to 252 days after onset of illness. In addition, IgM was detected from both CSF and sera of all four dengue patients with neural symptoms. The IgM titer in CSF (less than or equal to 1:20) was always lower than that in serum (greater than or equal to 1:80). Two cases with sequentially collected specimens showed the fading of IgM titer in CSF. As a matter of fact, it became undetectable about a month after onset of illness, which is apparently different from the situation in serum.