Herpes simplex virus subunit antibodies in patients with Parkinson's disease.

Serum IgG antibodies against herpes simplex virus (HSV) type 1 capsid, envelope, and excreted antigens in 52 patients with idiopathic Parkinson's disease, and in their age- and sex-matched controls, were assayed with a solid-phase radioimmunoassay. When compared with the controls, patients with Parkinson's disease were found to have a substantially increased antibody response against each of the HSV subunit antigens tested. The increased antibody response in patients with Parkinson's disease was not associated with the occurrence of recurrent HSV infections, since the difference in antibody levels was most evident when comparing patients without recurrent HSV infections with their respective control group. Consequently, the increased HSV antibody response in patient with Parkinson's disease might depend on some antigenic stimulation other than ordinary recurrent HSV infections, or alternatively, on the generally enhanced immunological reaction of the patients against HSV.

Comparison of enzyme-immunoassay and radioimmunoassay for detection of human rotaviruses and adenoviruses from stool specimens.

An enzyme-immunoassay (EIA) using polystyrene beads as the solid phase, guinea pig anti-rotavirus or anti-adenovirus immunoglobulin as primary antibody, rabbit anti-rotavirus or anti-adenovirus immunoglobulin as secondary antibody, and horseradish peroxidase-conjugated swine anti-rabbit immunoglobulin as indicator antibody, has been developed for the detection of human rotavirus and adenovirus antigens from stool specimens. A comparison of the developed EIA and radioimmunoassay (RIA) used previously in our laboratory was made with 250 stool specimens from children with acute gastroenteritis. Two specimens were found negative by both rotavirus and adenovirus EIAs but not by RIAs, but in each of these cases confirmatory EIA tests showed them to be false negatives. The confirmatory EIA tests were also necessary in several cases to prove the specificity of the binding or to eliminate non-specific reactions with specimens giving low positive reactions in EIA. The developed EIA was found to be as specific, sensitive and reliable as RIA in the routine diagnosis of rotavirus and adenovirus gastroenteritis provided that appropriate confirmatory tests were included.

Solid-phase enzyme-immunoassay for the detection of herpes simplex virus antigens in clinical specimens.

An indirect solid-phase enzyme-immunoassay (EIA) for the detection of herpes simplex virus (HSV) antigens in clinical specimens was developed. Rabbits and guinea pigs were hyperimmunized with highly purified nucleocapsids of HSV type 1. Microtitre plates were coated with 0.25 microgram of guinea pig anti-herpes simplex type 1 immunoglobulins per well. Clinical specimens, diluted in phosphate buffered saline containing fetal calf serum and detergents, were sonicated and incubated in the test wells overnight at 37 degrees C. Rabbit anti-HSV immunoglobulins were added as a secondary antibody at a concentration of 3.2 micrograms per well, and peroxidase conjugated swine antibodies against rabbit immunoglobulins, diluted 1:1,000, were used as a fourth layer. Clinical specimens which were sent for virus isolation or for isolation of Chlamydia trachomatis were tested by the developed assay and 20 out of 27 isolation positive specimens were found positive by EIA. Five out of 67 specimens negative by isolation gave positive results by EIA. The specificity of the results was confirmed by a control test using wells coated with normal guinea pig immunoglobulins. The test detected antigens from both serotypes of HSV. Cross reactions with varicella-zoster- or with cytomegalovirus were not found, and antigens from uninfected cells did not result in false positive results.

Detection of rubella virus antigen by one-step time-resolved fluoroimmunoassay and by enzyme immunoassay.

A one-step time-resolved fluoroimmunoassay (TR-FIA) and a conventional two-step enzyme immunoassay (EIA) for the detection of rubella virus antigen were developed. Two noncompetitive mouse monoclonal antibodies reactive with epitopes on the E1 polypeptide of rubella virus served as immunoreagents. One of the monoclones (7A6) was used for coating the solid phase, and the other (2C3) was labeled with either Europium chelate or with horseradish peroxidase. For TR-FIA, the specimen was incubated simultaneously with the label at 4 degrees C overnight. EIA required an overnight incubation with the specimen and after washing another 1 hr of incubation at 37 degrees C with the conjugate. The sensitivity of TR-FIA was 10 pg in an assay volume of 100 microliters, and the sensitivity of EIA was between 50 and 100 pg. Antigens could be detected by TR-FIA in supernatant of cultures of Vero cells 48 hr after inoculation with approximately 1 TCID50, while cytopathogenic effect (CPE) at that time was detected only in cultures inoculated with 10(5) TCID50 or more. Virus mixed with human amniotic fluid containing antirubella-specific IgG was detectable after an incubation at 37 degrees C for 5 days. The assays may find applications in prenatal diagnosis of intrauterine rubella infection, in early identification of viral antigens in cell culture and in monitoring production, concentration, and purification of rubella antigen for antibody assays.

Detection of influenza A virus by radioimmunoassay and enzyme-immunoassay from nasopharyngeal specimens.

Four-layer (indirect) radioimmunoassay (RIA) and enzyme-immunoassay (EIA) techniques were developed for the detection of influenza A and B virus in the sonicated nasopharyngeal specimens from patients hospitalized for acute respiratory infection. Polystyrene beads (RIA) or polystyrene microtiter plates (EIA) were used as the solid-phase, guinea pig antivirus immunoglobulins as the catching antibodies, rabbit antivirus immunoglobulins as the secondary antibodies, and 125I-labeled sheep antirabbit (RIA) or horseradish peroxidase conjugated swine antirabbit (EIA) immunoglobulins as the detector antibodies. A comparison of the developed RIAs and EIAs with the immunofluorescence (IF) method was made with 41 influenza A IF-positive and 150 influenza A IF-negative specimens. Each of the 41 influenza A IF-positive specimens was positive by the influenza A RIA and negative by the influenza B RIA. Out of 150 influenza A IF-negative specimens 3 specimens were found with weakly positive results in influenza A and B RIAs, but in each of these 3 specimens the binding proved nonspecific by the corresponding confirmatory tests. Using the EIa technique and the same immunoreagents as in RIA, identical results were obtained in each selected specimen tested. The developed RIAs and EIAs proved to be as specific and sensitive as the IF technique, and they should be practical in the diagnosis of respiratory infections directly from nasopharyngeal specimens.

Type-specific detection of parainfluenza viruses by enzyme-immunoassay and radioimmunoassay in nasopharyngeal specimens of patients with acute respiratory disease.

A four-year solid phase enzyme-immunoassay (EIA) and radioimmunoassay (RIA) techniques were applied for the type-specific detection of parainfluenza type 1, 2 and 3 virus antigens in sonicated nasopharyngeal specimens of patients with acute respiratory disease. Guinea-pig antiviral immunoglobulins as the secondary antibodies, and horseradish peroxidase-labelled swine anti-rabbit immunoglobulins (EIA), or 125I-labelled sheep anti-rabbit IgG (RIA) as the indicator antibodies. A total of 174 nasopharyngeal specimens collected by mucus extractor were tested, and the results were compared with those obtained by a routinely used immunofluorescence (IF) technique. The same number of positive specimens were achieved by the EIA and the RIA and 3/4, 4/4, and 19/20 immunofluorescence (IF)-positive nasopharyngeal specimens were positive by the parainfluenza type 1, 2 and 3 immunoassays respectively. In addition, four parainfluenza type 1 and three parainfluenza type 3 virus-positive specimens were found by the immunoassays out of 146 parainfluenza IF-negative specimens. The type-specificities of the parainfluenza immunoassays were confirmed by showing that no cross-reactions occurred when purified immunizing antigens and the EIA- and RIA-positive clinical specimens were cross-tested. The results indicate that parainfluenza type-specific antigens can be detected directly in nasopharyngeal specimens by the immunoassays and the preliminary findings with a small number of positive specimens suggest that these assays have a diagnostic potential which is similar or slightly better than the IF techniques.

Solid-phase radioimmunoassay of IgA, IgG, and IgM antibodies to human rotavirus.

A solid-phase radioimmunoassay (RIA) has been developed for the detection of human rotavirus-specific IgA, IgG, and IgM antibodies. Nebraska calf diarrhea virus grown in LLC-MK2 cell cultures in the presence of trypsin was directly adsorbed onto polystyrene balls, and antibodies that attached to the virus-coated balls were detected by subsequent binding of 125I-labeled antibodies specific to human alpha, gamma or mu chains of human Iga, IgG, or IgM immunoglobulins. A total of 116 serum specimens from 58 adult patients were tested. Binding ratios between the positive and the negative serum varied between 5 and 15, occasionally being 20 or more in the IgA and IgG assays, but rarely exceeding 3 in the IgM assay. The RIA was found to be more sensitive in detecting antibodies to rotavirus than the complement fixation (CF) test, the RIA titers obtained being 50--100 times as high as the CF titers. The method described offers a possibility of evaluating the immune response to human rotavirus and of detecting recent infection.

Comparison of four-layer radioimmunoassay and electron microscopy for detection of human rotavirus.

A four-layer radioimmunoassay (RIA) using polystyrene beads as the solid phase, anti-rota guinea pig IgG as primary antibody, anti-rota rabbit IgG as secondary antibody, and 125I-labelled sheep anti-rabbit immunoglobulin as indicator antibody has been developed for the detection of human rotavirus in stool specimens. A comparison was made of the developed RIA, routine electron microscopy, and research electron microscopy of 147 unconcentrated stool specimens from patients with infantile gastroenteritis. In routine electron microscopy 17 (11.6%) false-positive or false-negative results were obtained when compared with research electron microscopy. Each specimen positive in research electron microscopy was positive in RIA, and six additional RIA positives were found from 58 electron microscopy negative specimens. A confirmatory test was necessary to find out marginally positive but nonspecific reactions in RIA. The developed radioimmunoassay is slightly more sensitive than research electron microscopy of unconcentrated stool specimens and considerably more sensitive and more specific than routine electron microscopy.

Sensitive enzyme immunoassay for detecting immunoglobulin M antibodies to Sindbis virus and further evidence that Pogosta disease is caused by a western equine encephalitis complex virus.

An antibody capture enzyme immunoassay (EIA) was adapted for the detection of immunoglobulin M (IgM) antibody to Sindbis (SIN) virus. Sera from humans with a febrile illness characterized by rash and arthralgia in eastern Finland (Pogosta [POG] disease) and Sweden (Ockelbo disease) and from humans with western equine encephalitis (WEE) virus infection in the United States were tested for IgM antibodies by EIA. Seroconversions were documented in patients with POG disease and with WEE virus infections by using SIN virus as antigen and rabbit anti-SIN virus immunoglobulin; this confirms previous observations that POG disease is caused by a virus closely related to SIN virus and that IgM antibodies to WEE complex alphaviruses are not type specific. This IgM EIA provided a sensitive diagnostic and research tool applicable to epidemiologic problems posed by POG disease.

Comparison of monoclonal biotin-avidin enzyme immunoassay and monoclonal time-resolved fluoroimmunoassay in detection of respiratory virus antigens.

BACKGROUND: Detection of respiratory viruses by time-resolved fluoroimmunoassay based on monoclonal antibodies were developed in our laboratories in the late 1980s and they have been successfully used in daily diagnosis for more than seven years. Later, similar Biotin-EIAs were developed but the sensitivities were unsatisfactory. OBJECTIVES: Further optimization of monoclonal Biotin-EIAs and comparison of the optimized assays with TR-FIAs. STUDY DESIGN: Variations in test format, diluents, incubation times and temperatures, and different monoclonal antibodies were tested, and the final comparisons were made with TR-FIA using stored nasopharyngeal aspirates. RESULTS: The improvements in Biotin-EIA featured four changes which increased sensitivity in the assay: (a) test diluent contained diethylenetriamino-pentaacetic acid; (b) antigen and biotinylated detector antibody were added simultaneously; (c) reaction time was extended from 1 h at 37 degrees C to overnight at 4 degrees C; (d) from the thirteen monoclonal antibodies used in TR-FIA, ten were optimal also in Biotin-EIA, but in the parainfluenza 1 and 2 assays other monoclonals proved more sensitive. Out of 257 originally positive specimens tested in the comparison studies, 192 (74.7%) were again positive and 54 (21.0%) were negative in both assays; nine were negative in TR-FIA but positive in Biotin-EIA, while two specimens were negative in Biotin-EIA but positive in TR-FIA. The overall agreement between the two assays was 95.7%. CONCLUSIONS: All monoclonal Biotin-EIAs can be optimized to the same sensitivity as TR-FIAs for the detection of respiratory viruses. Laboratories which have no TR-FIA expertise may use Biotin-EIA in the diagnosis of acute respiratory infections.

Viral antibodies in the sera from patients with Parkinson disease.

An assay of antibodies to 15 various viruses and mycoplasma pneumoniae was performed on the serum specimens from 441 patients with Parkinson disease and from 443 healthy controls matched by sex, age, and place of residence, or from a representative group of these matched pairs. The main finding was a higher herpes simplex complement-fixing antibody level in patients with Parkinson disease than in controls. Patients with Parkinson disease had higher herpes simplex antibody titers more often than did their matched controls, and the matched controls, respectively, had low titers more often than the patients. The mean herpes simplex antibody titer (log2) of the patients (4.9) was significantly higher than that of controls (4.6) (p less than 0.01). This difference was also demonstrable when matched pairs were analysed for paired differences of herpes simplex antibody titers. For other viral CF and HI antibodies studied and for mycoplasma pneumoniae CF antibody, there were no significant differences either in the mean titers or in the distribution of individual titer values between the patients with Parkinson disease and the matched controls.

Virus antibody levels in serum specimens from patients with optic neuritis and from matched controls;.

Virus antibody levels in serum specimens taken in acute and convalescent phases from 77 patients with optic neuritits were tested by measles hamagglutination inhibition (HI), measles hemolysis inhibition (HLI), rubella HI, parainfluenza-1 HI, Epstein-Barr immunofluorescence (IF), and against 11 other viruses and mycoplasma pneumoniae with the complement fixation (CF) technique. The virus antibody levels were indicated to be usually very stable, and a fourfold change in virus antibody levels was demonstrated in only eight patients. The virus antibody levels were compared with specimens from two carefully selected control groups. The first control group consisted of 71 healthy persons matched in age, sex and place of residence with the patients with optic neuritis. The other control group consisted of 58 patients with various neurological diseases other than multiple sclerosis (MS) or infectious diseases of the central nervous system. The patients with optic neuritis had significantly higher measles antibody titres than the two control groups in both measles HI and measles HLI tests. Also in 33 patients with optic neuritis of unknown cause, the measles antibody levels were higher than in the control groups. On the other hand, various other antibody tests showed no statistically significant differences between patients with optic neuritis and the control group.

Virus antibody levels in the cerebrospinal fluid from patients with optic neuritis.

Virus antibody levels were studied in the cerebrospinal fluid (CSF) of 58 patients with optic neuritis and 58 control patients with no indication of multiple sclerosis (MS) or infectious disorders of the central nervous system (CNS). The specimens were tested against three different structural components of measles virus with measles hemagglutination inhibition (HI), measles hemolysis inhibition (HLI) and gel precipitation (GP) tests. Measles antibodies occurred in 62 per cent of CSF specimens from patients with optic neuritis, and 21 per cent of the controls. In the specimens from patients with optic neuritis, the positive rate figures were: for rubella HI test 35, parainfluenza-1 HI 16, and Epstein-Barr virus immunofuorescence (IF) 53 per cent. The frequencies in the control group were 10, 10 and 26 per cent, respectively. Serum/CSF antibody ratios below 80 occurred in measles tests in 45 per cent of patients with optic neuritis and 16 per cent of the control group. Some patients with optic neuritis (but none from the control group) had a reduced serum/CSF antibody ratio in more than one measles antibody test, The patients with optic neuritis had a higher frequency of low serum/CSF albumin ratios indicating blood brain barrier damage, There were, however, several patients with a normal serum/CSF albumin ratio but low serum/CSF immunoglobulin G and measles antibody ratios. This supports the hypothesis that local production of measles antibodies takes place in CNS in some patients with optic neuritis as well as in MS patients. The CSF specimens were further tested against 12 other viruses and mycoplasma pneumoniae complement fixation, but there were no positive specimens. New CSF specimens were taken from five patients during optic neuritis, and from seven patients later on during the follow-up because of the appearance of new neurological symptoms. There were no changes in virus antibody levels, except for two patients with an increase of measles virus antibody titres.

Preparation of La Crosse virus hemagglutinating antigen in BHK-21 suspension cell cultures.

Hemagglutinating and complement-fixing antigens of La Crosse virus (California arbovirus group) were produced in serum-free suspension cultures of BHK-21/13S cells. The appearance and production of these antigens were correlated with the titer of infectious virus. No significant differences in antigen titers were produced by varying virus dose 10-fold. Hemagglutinin appeared 6 to 8 hr after inoculation and reached peak titer in 14 to 22 hr. Both beta-propiolactone and Tween 80-ether treatment inactivated infectious virus in the antigens. Unlyophilized antigen was stable at -60, 5 and 24 C for at least 117 days but not for 1 year. Lyophilized antigen was stable for at least a year, however, at -20 and 5 C. Cell culture-produced antigen was more sensitive than brain-produced antigen in detecting hemagglutination inhibition antibody in human sera.

Coxsackie B virus antibodies in myocardial infarction.

Evidence for the association between Coxsackie B virus infections and myocardial infarction was studied in a prospective follow-up examination. Using the micro neutralization test, 9 (15%) of 59 patients with acute myocardial infarction and 1 (2.6%) of 38 control patients showed a fourfold, or higher, antibody increase in paired serum samples against Coxsackie B1-5 viruses. This difference is significant (p less than or equal to 0.05). None of the patients or controls revealed symptoms of a viral infection during the blood sampling. Virus isolation from throat and feces was negative in all patients and controls. This finding agrees with some previous studies suggesting that the Coxsackie B group may in some cases have a causal role in myocardial infarction, or may act as a triggering factor.

Time-resolved fluoroimmunoassay: a new test for rubella antibodies.

A new solid-phase immunoassay, time-resolved fluoroimmunoassay (TR-FIA), for rubella antibody was developed. The test used polystyrene beads coated with rubella antigen as the solid phase and a chelate of the rare earth metal europium as fluorescent label. A fast light pulse from a xenon flash lamp was used to excite the label, and after a 400-mus delay time the emission fluorescence was measured for 500-mus at 1-ms intervals during a total counting time of 1 s. Background fluorescence of short duration caused by fluorescent serum components and scattering could be eliminated by including the delay time. The TR-FIA was compared with hemagglutination inhibition, single radial hemolysis, and two types of radioimmunoassay (RIA) (a commercial RIA [GammaCoat] and a noncommercial RIA [T-RIA]) by using 60 serum specimens from patients with remote rubella infection. Overall agreement of TR-FIA with hemagglutination inhibition and GammaCoat was 96.7%, with single radial hemolysis 98.3%, and with T-RIA 100%. Linear regression coefficients varied from 0.83 to 0.94, the best being obtained with single radial hemolysis and T-RIA. TR-FIA was also found to be suitable for the diagnosis of acute infections, as significant increases of antibody level were detected in all 30 paired serum specimens tested from patients with an acute rubella infection. Sensitivity and specificity comparable to those of RIA and enzyme immunoassay were obtained with TR-FIA. Furthermore, the advantage that TR-FIA has over RIA is that it incorporates a nonisotopic and stable label; its advantage over EIA is that it is easier to standardize because no additional reaction with substrate is required.

Rotavirus, adenovirus, and non-viral enteropathogens in diarrhoea.

The aetiology of rotavirus and adenovirus in acute gastroenteritis was studied in a prospective series that comprised 283 children admitted consecutively with diarrhoea during a 1-year period. Rotavirus was associated in 49% of the cases by solid-phase radioimmunoassay and electron microscopical examination of stool specimens, or by serology. Adenovirus was detected by radioimmunoassay in the stool specimens of 29 (11%) patients, including 8 cases of possible dual infection with rotavirus. Rotavirus infections showed a typical age distribution and seasonal clustering between January and June, whereas the adenovirus-associated cases did not form a distinctive subgroup. Enteropathogenic bacteria were found in 10% of cases, and were nearly as common in association with rotavirus infection as not. Respiratory symptoms accompanied diarrhoea in 34% of the patients with rotavirus and in 25% of those with neither rotavirus nor adenovirus. Therefore we could not confirm the existence of a 'rotavirus syndrome', nor could we confirm an association of respiratory symptoms with rotavirus infection. Use of antibiotics before the onset of diarrhoea was more common among those with non-viral diarrhoea (23%) than in the rotavirus group (13%). Rotavirus infections appeared to be common among cases of 'antibiotic-induced' diarrhoea.

Detection of respiratory syncytial, parainfluenza type 2, and adenovirus antigens by radioimmunoassay and enzyme immunoassay on nasopharyngeal specimens from children with acute respiratory disease.

Four-layer antispecies radioimmunoassay (RIA) and enzyme immunoassay (EIA) procedures were developed for the detection of respiratory syncytial virus (RSV), parainfluenza type 2 virus, and adenovirus antigens in nasopharyngeal specimens from children hospitalized for acute respiratory disease. Polystyrene beads (RIA) or flat-bottomed polystyrene microtiter plates (EIA) were used as the solid phases, guinea pig anti-virus immunoglobulins were used as the captive antibodies, rabbit anti-virus immunoglobulins were used as the secondary antibodies, and 125I-labeled sheep anti-rabbit (RIA) or horseradish peroxidase-labeled swine anti-rabbit (EIA) immunoglobulins were used as the indicator antibodies. A comparison of the EIAs and RIAs with routinely used immunofluorescence (IF) techniques was made with 164 nasopharyngeal specimens collected from children with acute respiratory disease. Only 3 of 66 RSV IF-positive specimens were negative in RSV RIA, and of 83 RSV, parainfluenza type 2 virus, and adenovirus IF-negative specimens, 1 was positive in RSV RIA. Of 4 parainfluenza type 2 virus IF-positive and 11 adenovirus IF-positive specimens, each was positive in corresponding RIAs, and all 83 IF-negative specimens were negative in parainfluenza type 2 virus and adenovirus RIAs. The results of the RSV, parainfluenza type 2, and adenovirus EIAs confirmed the results of corresponding RIAs in each selected case tested. The RIAs and EIAs were found to be as specific and sensitive as IF techniques, and more practical in the rapid detection of respiratory viruses in nasopharyngeal secretions.