Enzyme-linked immunosorbent assay for detection of antibody to varicella-zoster virus.

Primary varicella-zoster virus (VZV) infection is a serious illness in immunocompromised individuals, and a rapid, sensitive, and reliable assay to identify high-risk VZV-susceptible patients would be clinically useful. An enzyme-linked immunosorbent assay (ELISA) for antibody to VZV was compared with the fluorescent antibody-to-membrane antigen (FAMA) assay and found to be similar in both sensitivity and specificity. The antibody titers determined by both assays were also similar. The absence of antibody detected by ELISA correlated with susceptibility to VZV infection. Because it is simple to perform and has equivalent sensitivity to FAMA, ELISA should be useful for VZV antibody testing in diagnostic and research laboratories.     

Enzyme-linked immunosorbent assay for detection of equine infectious anemia virus p26 antigen and antibody.

A sensitive specific enzyme-linked immunosorbent assay utilizing purified p26 antigen was developed for the detection of antibodies to equine infectious anemia virus in naturally and experimentally infected horses. Generally, antibodies to the virus could be detected by the enzyme-linked immunosorbent assay 3 to 4 days earlier than by the standard agar gel immunodiffusion test, and they could be detected more reliably in horses with weak or equivocal agar gel immunodiffusion test reactions. The enzyme-linked immunosorbent assay was also successfully applied to the detection of p26 antigen in tissue culture fluids.     

Enzyme-linked immunosorbent assay for immunoglobulin G antibody to encephalomyocarditis virus

An enzyme-linked immunosorbent assay for immunoglobulin G antibody to encephalomyocarditis virus was developed. This assay was comparable to antibody assay by neutralization. Its adaptability should be useful for laboratory and epidemiological studies of infections due to encephalomyocarditis virus.     

Enzyme-linked immunosorbent assay for detection of antibodies to murine hepatitis virus.

An enzyme-linked immunosorbent assay was developed for the detection of antibodies to murine hepatitis virus. A high prevalence of antibody to murine hepatitis virus was found by the enzyme-linked immunosorbent assay in colonies with a low prevalence of complement-fixing antibodies. Murine hepatitis virus strain A59 was found to be broadly reactive as an enzyme-linked immunosorbent assay antigen.     

Enzyme-linked immunosorbent assay compared with indirect immunofluorescence test for detection of Pneumocystis carinii specific immunoglobulins G, M, and A

An ELISA to measure Pneumocystis carinii-specific IgG, IgM, and IgA has been developed. The antigen was prepared from purified cysts by sonication and ultracentrifugation. Antigen particles with sedimentation coefficients less than 165 S were used. The technique has been compared with indirect immunofluorescence, using whole cysts as antigen. Ninety human sera were assayed. The results were significantly correlated. The ELISA-technique was more sensitive, and owing to the soluble antigen the daily variation was less than 1 per cent. The technique is useful for quick and reliable detection of Pneumocystis carinii antibodies in a routine laboratory.     

Virological and serological studies of Venezuelan equine encephalomyelitis in humans.

During the 1971 epidemic of Venezuelan equine encephalomyelitis (VEE) in south Texas, 203 suspect VEE cases were evaluated by the Center for Disease Control. Sixty-seven were confirmed as cases of VEE. Laboratory confirmation was accomplished by isolation of VEE virus from a serum specimen taken during the acute illness in 50 (75%) of the confirmed cases. Serological confirmation was obtained in 17 cases (25%). Virus isolations were most often obtained from sera collected during the first 3 days of illness. Peak serum virus titers (algebraic mean, 10(5-7) suckling mouse intracranial 50% lethal doses [SMICLD50] per ml) occurred on day 2 of illness. One-half of the sera from which virus was isolated contained at least 10(5) SMICLD50/ml, which has been shown to be sufficient to infect some vector mosquitoes. Blood from 13 virus-positive VEE cases was obtained 1 and 11 months after illness. Hemagglutination-inhibiting, complement-fixing, and neutralizing antibodies were formed by all 13 patients 1 month after illness. Hemagglutination-inhibiting antibody titers were essentially unchanged 11 months after illness. Complement-fixing antibody was undetectable 11 months after illness in 23% of cases and was detectable at dilutions of 1:8 or 1:6 in 77%. Neutralizing antibody (measured by log neutralization index) was not detectable 1 year after illness in one person (8%); titers had declined from 1.0 to 2.0 in 46%, were unchanged in 39%, and were not tested in one person (8%). No evidence of intrafamilial spread of VEE virus was obtained in either of two illness and antibody surveys. A randomized household illness and antibody survey of 681 Port Isabel residents revealed an inapparent infection ratio of 1:11 and an overall antibody prevalence of 3.2%.     

Detection of human immunoglobulins G and M antibodies to Rift Valley fever virus by enzyme-linked immunosorbent assay.

Rift Valley fever virus (RVFV) is an important human and animal pathogen in Africa and has been responsible for infections in travelers. Because of the aerosol infectivity and risk of dissemination of the virus, a need exists for simple, safe, serological tests for diagnosis. An enzyme-linked immunosorbent assay (ELISA) was developed to detect RVFV-specific immunoglobulins (immunoglobulin G [IgG] and IgM). In the test, a betapropiolactone-inactivated, sucrose-acetone-extracted, suckling mouse liver RVFV antigen was captured by mouse RVFV antibodies adsorbed to polystyrene plates. The test sample (human serum) was then added, and the binding of specific antibodies was indicated by alkaline phosphatase-conjugated swine anti-human IgG or IgM. A mu-capture IgM ELISA was also developed by using polystyrene plates coated with goat anti-human IgM incubated successively with serum sample, RVFV antigen, and indicator antibodies. The ELISA for RVFV-specific IgG proved to be more sensitive than hemagglutination inhibition or complement fixation tests and almost as sensitive as the plaque reduction neutralization test in detecting specific antibodies in human sera after vaccination. The two ELISA IgM tests could detect specific IgM antibodies during the first 6 weeks after RVFV vaccination. Three injections of inactivated vaccine were given on days 0, 6 to 8, and 32 to 34. ELISA IgM values for sera obtained on days 6 to 8 were negative or in the lower range of significance, on days 32 to 34 they were strongly positive, and on days 42 to 52 they were waning. Later sera were negative. The plague reduction neutralization test was negative on days 6 to 8 but rose progressively in later samples. These findings suggest that the three doses of RVFV vaccine induce a prolonged primary antibody response. The ELISA IgM could become an important tool for early diagnosis in acute human infection. A number of African sera, some of which were positive for RVFV by plaque reduction neutralization test, were also tested by ELISA IgG. There was good agreement between both tests.     

Enzyme-linked immunosorbent assay for detection of immunoglobulin G and M antibodies to teichoic acid in intravascular staphylococcal disease

An enzyme-linked immunosorbent assay (ELISA) for detection of IgG and IgM antibodies to cell-wall teichoic acids ofStaphylococcus aureus and three defined coagulase-negative staphylococci was tested using serum samples from 11 cases of intravascular coagulasenegative staphylococcal infections, 13 cases ofStaphylococcus aureus endocarditis, and 24 patients with no evidence of infection. IgG antibody titers to all four teichoic acids in the 13 patients withStaphylococcus aureus endocarditis were significantly different from those in noninfected control patients (p<0.0001). In contrast, IgG antibody titers in serum from 11 cases of intravascular coagulase-negative Staphylococcal infection were not significantly different from those in control sera. There were no differences in IgM antibody titers of the three groups. Although the ELISA was sensitive in detectingStaphylococcus aureus endocarditis, it was not reliable in the detection of intravascular coagulasenegative Staphylococcal infections, even when tested with specific teichoic acid.     

Detection of Lassa virus antigens and Lassa virus-specific immunoglobulins G and M by enzyme-linked immunosorbent assay.

Rapid diagnosis of Lassa fever is desirable for the timely therapeutic intervention and implementation of strict quarantine procedures both in West Africa field hospitals where the disease is endemic and at international crossroads. An enzyme-linked immunosorbent assay (ELISA) to measure Lassa virus antigens in viremic sera was developed in which experimentally infected monkeys were used as a model for the human disease. In this test, Lassa virus antigens in test sera were captured in wells of microtiter plates by monkey anti-Lassa virus immunoglobulin. Guinea pig anti-Lassa virus immunoglobulin was then added, and binding of specific immunoglobulin was quantitated by the addition of rabbit anti-guinea pig immunoglobulin followed by alkaline phosphatase-labeled anti-rabbit immunoglobulin. This test detected viremia titers as low as 2.1 log10 PFU/ml in experimentally infected monkey sera, a titer often exceeded in patients with Lassa fever. Inactivation of infectious virus by beta-propiolactone or gamma-irradiation did not diminish reactivity. Antigen-ELISA concentrations increased with infectivity for the first 10 days after infection but then declined while infectivity titers remained high, suggesting that the presence of humoral antibody in viremic sera diminishes the sensitivity of the antigen ELISA. Lassa virus-specific immunoglobulin M (IgM) titers measured in an IgM capture ELISA were detectable within 10 days of infection and peaked after 36 days but remained detectable for 1.5 years. The Lassa virus-specific IgG ELISA response was slightly delayed, peaking on day 73 but declining only slightly thereafter. These studies in a realistic primate model suggest that the antigen detection ELISA or the IgM capture ELISA described, in which beta-propiolactone-inactivated sera are used, should be useful for the rapid diagnosis of human Lassa fever.     

Effects of UV-irradiation upon Venezuelan equine encephalomyelitis virus.

Venezuelan equine encephalomyelitis virus labelled with [14C]aminoacids or [3H]uridine was purified and UV-irradiated. The irradiation led to the formation of uracil photodimers and the covalent linking of the nucleocapsid protein C to virion RNA. The inactivation of infectivity correlated with the formation of uracil dimers, whereas the RNA-protein links were formed at much higher doses of UV irradiation. The analysis of covalent RNA-protein complexes suggests that a fairly large fraction (at least one third) of the whole content of C protein is able to participate in the formation of UV-induced links, suggesting extensive contacts of RNA with protein with the nucleocapsid.     

Enzyme-linked immunosorbent assay for immunoglobulin G and M antibodies to Chlamydia trachomatis in human sera.

Microimmunofluorescence methods used for detection of immunoglobulin G and M antibodies to Chlamydia trachomatis are not available to many clinical laboratories. We evaluated a simple enzyme-linked immunosorbent assay in which serotype L2 elementary bodies are used as antigen. The enzyme-linked immunosorbent assay proved satisfactory for the detection of serum IgG. A total of 160 human sera were tested, and the results correlated well with those obtained by microimmunofluorescence. Results for IgM antibody detection were not as successful, and correlation with current methods was poor.     

Enzyme-linked immunosorbent assay for detection and typing of herpes simplex virus

An enzyme-linked immunosorbent assay for herpes simplex virus was tested using commercially available peroxidase-conjugated and unconjugated rabbit antibodies to herpes simplex virus type 1 and type 2 (DAKO immunoglobulins A/S, Copenhagen, Denmark). One hundred and thirty-seven clinical specimens from vesicles and superficial cutaneous lesions were tested and the results compared with virus isolation. In addition 210 herpes simplex virus isolates were typed. Forty-four specimens yielded herpes simplex virus both in the enzyme-linked immunosorbent assay and in tissue culture and 79 were negative in both tests. Fourteen were positive in isolation but negative in the enzyme-linked immunosorbent assay. Seventy-three isolates were typed as herpes simplex virus type 1 and 137 as herpes simplex virus type 2. The enzyme-linked immunosorbent assay was found to be rapid and easy to perform but less sensitive than conventional isolation in routine diagnostic work. For typing of isolates it was found to be a useful method for distinguishing herpes simplex virus type 1 and type 2.     

Enzyme-linked immunosorbent assay for detection of immunoglobulin M antibody to hepatitis B core antigen.

An enzyme-linked immunosorbent assay for detection of specific immunoglobulin M (IgM) antibodies against the core antigen of the hepatitis B virus (anti-HBc IgM) is described. The interference of IgM rheumatoid factor was evaluated quantitatively. In the anti-HBc IgM test, the rheumatoid factor gave false-positive results when the concentration exceeded 20 IU/ml. The rheumatoid-positive sera were disclosed by a control and retested for anti-HBc IgM after absorption of rheumatoid factor with latex particles aggregated with human IgG. In five of seven selected patients with acute hepatitis B followed to biochemical and clinical recovery, anti-HBc IgM was present transiently until antibodies against hepatitis B surface antigen (anti-HBs) appeared. Two patients had persistent anti-HBc IgM during the follow-up period. Four patients with hepatitis B surface antigenemia and progression to chronic liver disease did not clear their anti-HBc IgM in the period of observation (11 to 24 months). Anti-HBc IgM could not be demonstrated in 223 of 225 Danish blood donors. The two donors found positive for anti-HBc IgM also had anti-HBs. Twenty patients with acute A or non-A non-B hepatitis were negative for anti-HBc IgM. The enzyme-linked immunosorbent assay for anti-HBc IgM described here has a high specificity and sensitivity. The diagnostic relevance needs further evaluation, including quantitation of anti-HBc IgM, but the results presented indicate that anti-HBc IgM may be helpful in differentiating between prior and recent or ongoing hepatitis B infection.     

The immunity generated in white rats vaccinated with strains of the Venezuelan equine encephalomyelitis virus]

The existence of virulence gradient in the main members of Venezuelan equine encephalomyelitis virus complex (VEE) was established by subcutaneous inoculation of immunologically competent outbred rats weighing 50-70 g. The virulence of the strains may be judged by a parameter such as the weight of the animal in 5 or 10 days after inoculation. The highest degree of protection was observed in the animals immunized with strain 15, the least in those immunized with strain 230. The above results indicate that adult white rats may be used as a model for the evaluation of the efficacy of protective preparations against viruses of the VEE complex.     

Properties of virions and ribonucleoproteins of Venezuelan equine encephalomyelitis virus.

The physicochemical properties of Venezuelan equine encephalomyelitis (VEE) virus and of its ribonucleoprotein (RNP) were studied. Upon purification in a discontinuous or linear sucrose gradient, the losses of infectivity were small (25%), and 96% of cellular proteins were removed. The purified virus was homogeneous with respect of sedimentation rate (s20, w = 265 S). The CsCl gradient was unsuitable for purification of infectious virus because the latter was destroyed at high CsCl concentrations. Buoyant density of the virus in CsCl after formaldehyde fixation was 1.21--1.22 g/cm3. Treatment of the virus with 1% Nonidet P-40 proved to be the most effective method for isolation of the RNP. The structures thus obtained contained practically all of the viral RNA and about 20% of viral proteins, and were homogeneous with respect of sedimentation rate (153 S) and buoyant density (1.40--1.42 g/cm3 in CsCl after formaldehyde fixation). The RNPs were sensitive to ribonuclease.     

Enzyme-linked immunosorbent assay for evaluation of immunity to measles virus.

An enzyme-linked immunosorbent assay for the determination of immunity to measles virus was developed and standardized; it was compared to the hemagglutination inhibition and plaque reduction neutralization methods for sensitivity and specificity. The conditions of the enzyme-linked immunosorbent assay were adjusted such that groups of susceptible and immune individuals were clearly separable on the basis of the reactivity of a single (1:100) dilution of their sera to viral and control antigens. The range of values corresponding to susceptibility and immunity was defined by using the distribution of values observed from testing sera obtained from susceptible and immune control groups. The enzyme-linked immunosorbent assay was then applied in a study of measles vaccinees and found to be more sensitive than the hemagglutination inhibition method and equal in sensitivity to the plaque reduction neutralization method. The three methods were equal in specificity. Thus, the measles virus enzyme-linked immunosorbent assay is a rapid, reproducible, sensitive, and specific method for screening for the presence of measles antibody.