Methods for screening the naturally acquired and vaccine-induced immunity to the mumps virus

Since the introduction of the measles, mumps and rubella (MMR) vaccine in Sweden in 1982, a yearly evaluation of the immunity pattern and seroconversion rate of 12-year-old children has been carried out. To be able to realize large-scale, follow-up studies, techniques have to be used which are not too labour-intensive and time-consuming but are sensitive enough to detect past immunity and post-vaccination titres. We report tests with haemolysis-in-gel (HIG) technique and an enzyme-linked, immunosorbent assay (ELISA) compared with neutralization (NT) for the detection of mumps antibodies. This study comprises 226 paired serum samples obtained between 1985 and 1989. One hundred and forty-one of the paired samples had been selected because they had given negative or borderline readings, using HIG technique. The remaining samples were consecutive pre- and post-vaccination sera obtained in 1989 from 85 vaccinees from one area in Sweden. HIG technique gave both false positive and false negative readings, compared with NT as also the false positive sera were detected. Non-inactivated sera could not be used in the NT test against mumps virus, owing to unspecific NT reactions. No differences between non-inactivated and inactivated sera by NT were seen, as against other paramyxoviruses. Cross-reactivity between mumps and parainfluenzae in NT tests was not demonstrated. The ELISA test proved more reliable and specific than HIG and was more sensitive than NT. Some post-vaccination sera from vaccinees who failed to seroconvert by NT contained high levels of mumps antibody detectable by the ELISA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protection of mumps in children with various underlying diseases: application of a live attenuated mumps and trivalent measles-rubella-mumps (MRM) vaccines in these children

A live attenuated mumps and trivalent measles-rubella-mumps (MRM) vaccines have been applied to 887 and 148 children with various underlying diseases at the vaccine clinic of Osaka University Hospital between 1975 and 1985, respectively. Clinical reactions after mumps vaccination occurred in only 7 children (0.8%) and those after MRM vaccination in 28 children (19%), but their underlying diseases were not deteriorated by either vaccination. Clinical follow up study revealed that 2 of the 430 children immunized with mumps vaccine had contracted the disease during 7 year period and 2 of the 123 children immunized with MRM vaccine had contracted clinical mumps or rubella during 3 year period. The seroconversion rates after mumps vaccination were 70% and 61% by the hemagglutination inhibition (HI) test and neutralization (NT) test, respectively, while 94% by the fluorescent antibody to membrane antigen (FAMA) test. Those after MRM vaccination were 87% for measles, 96% for rubella by the HI test and 89% for mumps by the FAMA test. Serological follow up study revealed that antibodies elicited by mumps vaccination were sustained without substantial decline for at least 7 years. These results suggest that a live attenuated mumps and MRM vaccines are safe and effective in children with various underlying diseases.     

Antibody response in school children to live rubella vaccine (Cendehill strain)

The efficacy of an attenuated rubella virus vaccine, Cendevax, was tested on 65 school children. Forty-nine of them (75%) had pre-existing antibodies and in these there was no increase in the HAI antibody titres after administration of the vaccine. Sixteen children (25%) had no demonstrable rubella HAI antibody prior to vaccination. From the latter group, postvaccination serum samples were available from only 11, and 10 of these seronegative children showed seroconversion after vaccination. The geometric mean HAI titre was 1:180. Seven of the 10 postvaccination serum samples had complement-fixing antibodies and specific IgM antibodies were detected by the immunofluorescence test in 8. No correlation was observed between the CF and the IgM antibodies.     

Methods for screening the naturally acquired and vaccine-induced immunity to the measles virus

Since the measles, mumps and rubella (MMR) vaccine was introduced into Sweden in 1982, a yearly evaluation of the immunity patterns and sero-conversion rates in 12-year-old children has been carried out. Since 1977, about half of the pre-school children have been vaccinated against measles. This study includes two study groups. (1) 145 selected pre- and post-vaccination samples tested by the haemolysis-in-gel (HIG) technique and the neutralization test (NT). The selection was made from 1298 12-year-old schoolchildren in 1986 and 1987, whose pre-vaccination sera had shown negative or borderline reactions to the HIG technique. (2) Consecutive pre- and post-vaccination samples obtained from 190 vaccinees in 1988 and 1989. These samples were studied by an enzyme-linked, immunosorbent assay (ELISA) and compared to the NT. The NT and the HIG tests yielded congruent results in early post-vaccination sera from children susceptible to measles prior to vaccination. In late post-vaccination samples, the NT and the HIG tests were discordant, up to 25% of the NT-positive samples being negative by the HIG technique. In no instance did the ELISA produce discrepant results, compared with those of the NT. With both this assays significantly lower antibody levels were detected in late post-vaccination sera (8-11 years) compared to early post-vaccination samples (P less than 0.001) or to sera obtained after natural infection.     

Detection of serum antibodies against Chlamydia pneumoniae by ELISA

Abstract Chlamydia pneumoniae causes pneumonia and other respiratory infections in children, adolescents and adults. We tried to evaluate the diagnostic value of detection of serum antibodies by ELISA for C. pneumoniae infections in Japanese children. Serum IgG, IgA and IgM antibodies to C. pneumoniae were determined by the microimmunofluorescence (MIF) test. Serum IgG and IgA antibodies were also determined by ELISA test kits. Results obtained by ELISA were compared with those obtained by MIF test. IgG antibody to C. pneumoniae was detected in 135 (39.5%) by ELISA and in 125 (36.5%) by MIF out of 342 sera from Japanese infants and children without respiratory infections (aged from 2 months old to 15 years old). IgA antibody to C. pneumoniae was detected in 129 (37.7%) by ELISA and in 117 (34.2%) by MIF out of 342 sera tested. Of 342 specimens 113 were IgG-positive by ELISA and MIF (sensitivity: 90.4%, specificity: 89.9%, r = 0.853). Of 342 sera 28 had IgG antibody titers of 1:256 and none had titers 1:512 or higher by MIF. Of 28 infants and children a total of nine were less than 4 years of age. On the other hand, of 342 specimens 99 were IgA-positive by ELISA and MIF (sensitivity: 84.6%, specificity: 86.7%, r = 0.769). Of 342 sera 16 had IgA antibody titers of 1:256 or higher by MIF. Of 16 infants and children, ten were less than 4 years of age. ELISA had excellent sensitivity and specificity relative to MIF test for detection of IgC and IgA antibodies to C. pneumoniae . It was suggested that C. pneumoniae infection in Japanese infants and children under 4 years of age was not infrequent.     

Comparison of 4 serological tests--complement fixation, neutralization, fluorescent antibody to membrane antigen and immune adherence hemagglutination--for assay of antibody to varicella-zoster (V-Z) virus.

Four tests for antibody to varicella-zoster (V-Z) virus were compared; these were tests of complement fixation (CF), neutralization (NT), fluorescent antibody to membrane antigen (FAMA) and immune adherence hemagglutination (IAHA). Fifty-two sera from patients with varicella and zoster and from recipients of live varicella vaccine were examined by the 4 tests. The CF test was least sensitive, but the antibody titers by the NT, FAMA and IAHA tests were roughly comparable. The IAHA test was the simplest and fastest to perform, and appeared suitable for routine serological assay to V-Z virus. The correlation between the IAHA antibody titer and susceptibility of individuals to clinical varicella was investigated retrospectively using sera obtained during 2 outbreaks of varicella in an institution for children, where all the unvaccinated children had developed varicella symptoms. Most of the 25 pre-exposure sera from unvaccinated children examined by the IAHA test had tiers of less than 1:2. In contrast, all the 23 sera from vaccinated children who did not develop varicella had detectable antibody titers of 1:2 to 1:64. These results indicate that the IAHA titer reflects the susceptibility or resistance of individuals to clinical varicella.     

Detection of Coronavirus 229E Antibody by Indirect Hemagglutination

Tannic-acid treated sheep erythrocytes (fresh or glutaraldehyde preserved) were sensitized with 229E antigens from human embryonic lung (RU-1) cell cultures. Indirect hemagglutination (IHA) antigen titers in 229E-infected cell cultures paralleled virus infectivity and complement fixation (CF) antigen titers. The identity of the IHA antigen was confirmed by testing extracts from inoculated and control cell cultures for ability to inhibit IHA. Also, significant increases in IHA antibody were demonstrated with acute and convalescent serum pairs from patients with proven 229E infections. A comparison of IHA, neutralization and CF titers for 229E antibodies was made on human sera drawn from different populations. The IHA and neutralization results were in agreement on 93% of the 129 sera found to be positive by at least one of three tests. The number of antibody titers detected by the CF test was insufficient to permit comparison. Hyperimmune sera from animals immunized with OC 43 did not react with 229E by IHA. Also no increase in IHA antibody was demonstrated with acute and convalescent serum pairs from patients with seroconversions to OC 43. These findings suggest that the IHA test provides (i) a rapid and sensitive method for serodiagnosis of 229E infections and (ii) a simple and inexpensive method for seroepidemiological studies.     

Evaluation of the usefulness of the ELISA method for determining the level of enterovirus antibodies in serum and cerebrospinal fluid

The usefulness of the methods was compared: complement fixation test (CFT), neutralization test (NT) and ELISA IgG and IgM against enteroviruses for the evaluation of specific immune reaction in sera and cerebrospinal fluid (CSF) samples of patients with confirmed enterovirus infections. The criteria were established for the assessment of ELISA results in rapid diagnosis of enterovirus neuroinfections. The criteria accepted by the producer lowered the sensitivity of the method and the possibility of recognition of local synthesis of antibodies in the CNS. The use of serum negative in CFT and negative CSF as reference for the determination made possible using of that kit for rapid diagnosis of neuroinfections. The modified ELISA IgG test makes possible determination of antibodies in CSF and serum, and accepting the generally recognized criteria for local production of antibodies in the CNS the ELISA test makes possible rapid diagnosis of neuroinfections which is not possible by other methods.     

A shortened dengue IgM capture ELISA using simultaneous incubation of antigen and peroxidase-labeled monoclonal antibody

A shortened IgM capture ELISA for the detection of dengue IgM antibodies using simultaneous incubation of antigen and peroxidase-labeled monoclonal antibody was described. The shortened two-step assay was compared with the four-step IgM capture ELISA on sera from dengue patients confirmed by the hemagglutination inhibition (HI) test. When paired acute and convalescent sera were tested, the shortened ELISA showed 100% agreement with HI results. It detected dengue IgM antibodies in the acute sera of 66% of patients with a primary dengue infection, 60% of patients with a secondary infection, and 98% of patients with a presumptive secondary infection. When the results of 151 dengue patients were combined, 75% of the acute sera were positive by the shortened IgM capture ELISA.     

Indirect haemagglutination (IHA) test in the serodiagnosis of amoebiasis

Among 72 patients clinically suspected of Entamoeba histolytica (E. histolytica) infections, 39 positive cases (54%) were detected serologically by the indirect hemagglutination (IHA) test. Parasitologically, microscopic examination of three consecutive stool specimens from all these patients indicated positivity for E. histolytica cysts and or trophozoites in 10 of the patients with IHA antibody titers greater than or equal to 1:128, which is of clinical significance. Another 2 patients were parasitologically positive but showed low IHA antibody titres (1:32-1:64); follow up indicated response to treatment with metronidazole. The highest serological positivity (100%) were detected in patients with liver abscess, all were clinically proven cases of extra-intestinal amoebiasis. IHA antibody levels of clinical significance were seen in all four patients with chronic dysentery with parasitological evidence of E. histolytica in their stools. In a group of patients with abdominal pain nine positives were detected serologically, four of which were positively diagnosed concurrently by parasitology; the remaining five patient's sera showed high IHA antibody titres with absence of cysts or trophozoites in stools, indicative possibly of persistence of antibodies from past infection. The serologic determination of E. histolytica IHA antibodies in a control group consisting of normal healthy school children and adults of both sexes without any clinical evidence of amoebiasis showed the absence of any positive titres of clinical significance; low titres (1:32-1:64) were detected in 5.2% of 232 sera tested. Parasitological examination of three consecutive stool specimens from all individuals in the control group showed the presence of cysts of E. histolytica in just two among 232 tested (0.9%).     

Impact of ELISA and immunoblot as diagnostic tools one year after eradication of Helicobacter pylori in a multicentre treatment study

The performance of serological tests for Helicobacter pylori infections is hampered by the persistence of antibodies after eradication therapy or spontaneous healing. Detection of different antigens or immunoglobulin classes might have an impact on the validity of serodiagnosis. The aim of this study was to assess the decrease in IgA and IgG antibody levels after eradication of H. pylori. Serum samples of 242 patients with active duodenal ulcer were tested with the ELISA and the immunoblot (IB) techniques for H. pylori-specific IgA and IgG antibodies before therapy and 1 year after successful eradication. From a total of 81 patients paired sera were available. At the end of the follow-up period ELISA antibody titres from the IgA class had decreased from a mean value of 6.69 to 4.26 units (P = 0.0001), and IgG class antibody titres from a mean value of 21.9 to 12.1 units (P = 0.0001). Regarding seroreversion, from 34 initially IgA positive sera 16 (47%), and from 74 IgG positive sera 18 (24%), had definitively reverted to 'negative'. One year after eradication, when tested with the immunoblot, the antibody responses against specific antigens of 37% IgA-positive sera (23/62) and 8% IgG-positive sera (6/78) reverted to 'negative', compared to a seroreversion rate of 27% of the anti-CagA IgA-positive sera (18/67) and of 9% of the anti-CagA IgG-positive sera (7/79). In conclusion, despite an overall significant decrease of H. pylori antibodies, both tests cannot be recommended for monitoring treatment success.     

ELISA for detection of IgM and IgG antibodies to sandfly fever sicilian virus

• •An enzyme-linked immunosorbent assay (ELISA) was developed to detect specific human immunoglobulin G and M antibodies to sandfly fever Sicilian (SFS) virus. Acute and early convalescent serum pairs with ⩾ 7 days between the 2 specimens were available from 20 patients and all showed significant optical density (OD) increase and significant titre rise (⩾ 4-fold) by IgG ELISA. However, negative or borderline-positive sera were found as late as 11 days after onset of symptoms when tested by IgG ELISA.
• •Specific IgM antibodies were detected during the first week of symptoms, and maximum OD values were obtained during the first 4 weeks after onset of disease. The IgM OD values declined over the following 3–9 months. All sera collected later than 14 months post-onset were negative by IgM ELISA.
• •The combination of early antibody response and the need to test only one serum specimen gives IgM ELISA an advantage over IgG ELISA in patient diagnosis.
• •The IgG ELISA was also evaluated as a seroepidemiological tool and compared to a plaque reduction neutralization test (PRNT) using sera from a normal Cypriot population. Of 183 sera tested, 34 (19%) were positive in plaque reduction neutralization tests (PRNT) and 113 (62%) by IgG ELISA. A number of PRNT-negative sera were strongly positive by IgG ELISA and also by indirect immunofluorescence test, which may suggest the presence of a virus related to SFS in Cyprus which has not yet been isolated.

     

The effect on kaolin adsorption of serum on the virus neutralization and enzyme-linked immunosorbent assays of antibody to bovine herpesvirus 1

Two procedures have been used for measuring antibody titres to bovine herpes virus 1 (BHV1): the serum neutralization (SN) test and enzyme-linked immunosorbent assay (ELISA). One hundred and thirty-two sera selected for their low SN titres were tested both unadsorbed and after adsorption with kaolin to determine the effect of kaolin on the titres. With ELISA, the titres of unadsorbed and kaolin adsorbed were not significantly different but with the SN test many treated sera, originally with weak positive titres, became negative after kaolin adsorption. Thus, if the ELISA results are specific for BHV1 antibody then the SN test findings suggest that treatment of sera with kaolin, rather than removing a viral inhibitor, removes a substance from the serum which potentiates SN antibody. This in turn indicates that low SN titres (reciprocal of titre less than or equal to 4, for instance) are probably specific for BHV1 SN antibody whether or not they are abolished by kaolin treatment of the serum.     

Single-serum diagnosis of recent rubella infection with the use of hemagglutination inhibition test and enzyme-linked immunosorbent assays

Single-serum diagnosis of recent rubella infection was attempted with the use of hemagglutination inhibition (HI) test and two commercially available enzyme-linked immunosorbent assays (ELISAs). The period during which IgM antibody was detectable by IgM capture ELISA was 4 to 30 days after the onset of rash. Rubella IgG ELISA values relative to HI titer were lower in the sera from the patients with recent infection than in the sera from the subjects with remote infection. IgM ELISA and the combined use of IgG ELISA and HI test are two useful methods of single-serum diagnosis of recent rubella infection.     

Development of a solid-phase immunoenzyme method for determining the antibody level in the blood sera of persons vaccinated with influenza vaccine

To control the effectiveness of vaccination against influenza, the optimum conditions for making the enzyme-linked immunosorbent assay (ELISA) with a view to determine the level of anti-influenza antibodies in human blood sera have been established. The kinetics of influenza virus adsorption in the wells of ELISA polystyrene plates and the kinetics of the interaction between the immobilized antigen and species-specific peroxidase-labeled antibodies have been studied. The method has been shown to be more sensitive than the hemagglutination inhibition test in the determination of seroconversion in persons immunized with influenza vaccine.     

流行性出血热病毒单克隆抗体的特性鉴定及其对病毒结构蛋白作用的初步研究

本文运用放射免疫沉淀法(RIP)、Western-blot及ELSA夹心法,对一组针对流行性出血热病毒(EHFV)L99株、C4株的单克隆抗体(McAb)进行特性鉴定,其中4株McAb针对糖蛋白G2,29株针对核壳蛋白(NP),1株针对糖蛋白G1和NP。微量中和试验和血凝抑制(HI)试验分析表明,虽绝大多数抗NP McAb既无中和活性亦无HI活性,但有1株例外。具有明显的中和活性和HI活性,提示EHFV核壳蛋白上可能存在中和抗原和血凝抗原决定簇。2株抗G2 McAb具有较高的HI活性,1株抗G2 McAb有较低的中和活性和较高的HI活性,另一株抗G2 McAb仅具有中和活性,表明EHFV糖蛋白G2上存在独立的中和与血凝抗原决定簇,也可能存在具有中和、血凝双重功能的决定簇。竞争ELISA分析显示。G2蛋白上某些中和位点和血凝位点虽然独立分布,但在某一区域可能相当靠近或有部分重叠。     

Characterization of anti-Citrobacter 036 specific polysaccharide monoclonal antibodies

Monoclonal mouse antibodies specific for the 0 antigen of Citrobacter 036, a homopolymer of beta (1----2)-linked 4-deoxy-D-arabinohexose, were generated by the hybridoma technique. Balb/c mice were immunized with killed whole-cell vaccine and initial selection of active clones was based on enzyme-linked immunosorbent assay (ELISA) employing purified lipopolysaccharide (LPS). Concentrated culture supernatants from selected hybrid cultures were used to identify 10 0-antigen specific monoclonal antibodies using the multiple criteria of immunoprecipitation of 0 chains and LPS, inhibition by acid hydrolyzed 0 chains in the screening ELISA, and antibody class analysis. Four monoclonal antibodies were chosen for further study using dose-dependent 0-chain inhibition of ELISA and passive hemagglutination, passive hemolysis, and bacterial agglutination titres. When screened with Citrobacter serotypes known to contain the sugar 4-deoxy-D-arabinose, passive hemagglutination tests showed that the two monoclonal antibodies examined possessed titres which could be correlated with the reported 4-deoxy-D-arabinohexose content of the respective LPS's. This sugar is an antigenically important unit of several Citrobacter serotypes as defined by these well-characterized monoclonal antibodies.     

IgG avidity ELISA test for diagnosis of acute toxoplasmosis in humans

Serum samples, 100 in the total number, were collected from different laboratories in Tehran, Iran and tested for anti-Toxoplasma specific IgG and IgM antibodies using indirect immunofluorescent antibody test (IFAT). Using the IgG (chronic) and IgM (acute) positive samples, the IgG avidity test was performed by ELISA in duplicate rows of 96-well microtiter plates. One row was washed with 6 M urea and the other with PBS (pH 7.2), then the avidity index (AI) was calculated. Sixteen out of 18 (88.9%) sera with acute toxoplasmosis showed low avidity levels (AI ≤ 50), and 76 out of 82 (92.7%) sera in chronic phase of infection showed high avidity index (AI>60). Six sera had borderline ranges of AI. The results showed that the IgG avidity test by ELISA could distinguish the acute and chronic stages of toxoplasmosis in humans.     

Diagnostic use of ELISA, IgG, IgM, IgA and ELISA IgG avidity in recent and chronic toxoplasmosis]

Toxoplasmosis, a world-wide zoonotic infection, is generally asymptomatic and benign in immunocompetent individuals, but it can be serious in immunodeficiencies particularly in patients with acquired immunodeficiency syndrome and in children infected in utero. So, it is important to dispose methods which permit discriminate between recent and chronic infections. In order to contribute to improve the diagnosis of toxoplasmosis ELISA IgG, IgM, IgA and ELISA IgG avidity were performed in 15 and 24 sera from patients suspected of having acute and chronic infection respectively, according dye test (DT) titres. ELISA IgG was positive in both groups, ELISA IgM was positive in 78.6 and 58.3% respectively, while ELISA IgA was positive in 85.7 and 33.3% of recent and chronic group respectively. In those sera with low IgG avidity (18.8%) we found specific IgM in 71.5 and 4.2% and IgA in 78.6 and 0.0% of recent and chronic groups respectively. Parallelling, 208 sera samples were classified according to the results of DT, indirect hemagglutination and complement fixation tests in the following groups: acute (97), intermediate (36), chronic (35) and negative (40). The results were: acute (96.9-64.9-55.6 and 65.9%); intermediate (97.2-63.8-44.4 and 47.2%); chronic (45.7-42.8-5.7 and 34.3%) for IgG, IgM, IgA and low IgG avidity respectively. The use of both acute markers, IgA and low IgG avidity in the diagnosis of toxoplasmosis is discussed.