Ilkka Julkunen

Enzyme immunoassay, complement fixation and hemagglutination inhibition tests in the diagnosis of influenza A and B virus infections. Purified hemagglutinin in subtype-specific diagnosis

The efficacy of enzyme immunoassay (EIA) in detecting diagnostic antibody rises to influenza A and B viruses was compared with complement fixation (CF) and hemagglutination inhibition (HI) tests in 455 patients with an acute respiratory infection. EIA and HI detected significantly more diagnostic antibody rises against influenza A than the CF method (96 and 87 vs. 47, respectively). In the case of influenza B significantly more diagnostic influenza B antibody rises were observed by EIA than by CF or HI (59 vs. 37 and 40, respectively). In most of the cases antibody rises in EIA were found in both IgG and IgA isotypes whereas increases in IgM antibodies were seen less frequently. Purified hemagglutinins (HA) were prepared from influenza A HI- and H3-subtypes and from influenza B viruses and used as antigens in EIA and the results were compared with those of HI. Infections caused by influenza A HI-subtype showed good homologous antibody responses in EIA but heterologous antibody responses to H3-subtype and influenza B HAs were frequently observed. Heterologous responses were clearly less frequent in patients with infections caused by the H3-subtype. Influenza B infections occasionally raised HA antibodies against influenza A H1-subtype but not to the H3-subtype. Interestingly, HI detected these heterologous responses at least as frequently as EIA. When whole viruses were used as antigens in EIA, subtype specificity was not observed and cross-reactions between influenza A and B virus antibodies were found. These observations suggest that, although EIA can show greater diagnostic efficacy over HI and CF methods, HI is still the serological method of choice in determining the causative subtype of influenza A virus infection.

CHRONIC ENCEPHALOMYELITIS WITH SPECIFIC INCREASE IN INTRATHECAL MUMPS ANTIBODIES

n Abstractn n Symptoms of severe encephalomyelitis developed in a 31-year-old man in 1967. He had a high serum antibody titre to mumps virus associated with a polymorphic cell reaction and an increased protein concentration in cerebrospinal fluid (CSF). He recovered considerably within a year and was able to resume work. In 1975 his condition deteriorated again; it improved during the following few years, but a further deterioration then occurred. In March, 1981, the complement-fixing antibody titre to mumps virus was 1/32 in the serum and 1/4 in the CSF. In November, 1981, the CSF IgG index was increased and the altered serum/CSF antibody ratio persisted. The specificity of the altered antibody ratio was confirmed by the single radial haemolysis test and an immunoassay specific for mumps virus. Antibodies against the mumps virus envelope glycoprotein, M-protein, and nucleoprotein could be demonstrated by immunoprecipitation and the antibody patterns in serum and CSF were similar. Antibodies against other microorganisms were not detected in the patients CSF, and mumps antibodies were not found in the CSF specimens of 57 control patients. This case may be an example of a new disease—chronic mumps virus infection in the central nervous system.n n

Serological diagnosis of influenza A and B infections by enzyme immunoassay. Comparison with the complement fixation test

Paired sera from 784 patients with symptoms of acute respiratory disease were examined for antibodies against influenza A, B and parainfluenza (1 and 3) viruses by complement fixation (CF) and enzyme immunoassay (EIA). The internal variation of the EIA test results was low and an increase of 0.250 in absorbance values which corresponded to a two-fold increase in end-point titres was considered a diagnostic antibody rise. EIA detected significantly more diagnostic rises than the CF test in the case of influenza A (222 vs. 162, P less than 0.001) and parainfluenza virus antibodies (29 vs. 16, P less than 0.01). More diagnostic rises in influenza B antibodies were also observed by EIA compared to the CF test (104 vs. 99, not significant). There were only two patients who showed a diagnostic rise in CF antibodies (both influenza B) but not in EIA. Most often patients with a diagnostic antibody rise only by the EIA method had a two-fold rise in the respective CF antibodies (68% of cases). EIA was found to be a sensitive and reliable method for the serological diagnosis of influenza A, B and parainfluenza infections.

Chronic mumps virus encephalitis: Mumps antibody levels in cerebrospinal fluid

To study the outcome of mumps virus encephalitis 47 patients were contacted 1-15 years after the acute encephalitis associated with mumps virus infection. Twenty-three patients experienced clinical sequelae such as difficulties in memory and learning, focal motor or sensory signs, and loss of hearing and visual acuity. Lumbar puncture was performed on 8 patients. Antibodies to mumps virus were detected in 6 cerebrospinal fluid (CSF) specimens using enzyme immunoassay and in 3 patients an abnormal serum/CSF antibody ratio was observed 11, 26 and 58 (controls greater than 85); 14.3, 1.4 and 6.1 years after the acute encephalitis, respectively. Antibodies to other microbes were either undetectable in the CSF or the serum/CSF ratios were normal. The clinical sequelae in about half of the patients and the signs of intrathecal mumps antibody production are suggestive of a chronic process in the central nervous system after encephalitis associated with mumps virus infection.

Proportions of immunoglobulin isotypes in paralytic poliomyelitis and after vaccination

Immunoglobulin isotype composition of poliovirus antibodies was studied by isotype-specific solid-phase radio-immunoassay (RIA) in four patients with paralytic poliomyelitis, five adults receiving live poliovirus vaccine as a booster immunization, and seven children receiving first doses of inactivated poliovirus vaccine. In paralytic poliomyelitis serum and cerebrospinal flind (CSF) poliovirus antibodies were mainly of IgG1, IgG3, and IgA isotypes. IgM antibodies were found in sera but not in CSF. Either IgG2 and IgG4 antibodies were undetectable or the titers were low. In adults who had received live trivalent poliovirus vaccine, antibodies against poliovirus type 3 were detected in IgG1 (53% of total antibodies), IgG3 (25%), IgM (9%), IgA (8%), IgG2 (3%), and IgG4 (2%) isotypes. In prevaccination and late postvaccination sera the share of IgG3 antibodies was exceptionally high (35%). In children who received inactivated poliovirus vaccine, antibodies developed in IgG1 (53–61% of total antibodies for poliovirus types 1, 2, and 3), IgG3 (12–21%), and IgM (23–33%) isotypes. Antibody levels in IgG2, IgG4, and IgA isotypes were low and observed only in a few cases. Like other viral antibodies IgG1 and IgG3 isotypes were the major IgG subclasses in poliovirus antibodies.

Preparation of nasopharyngeal secretions for immunofluorescence by one-step centrifugation through Percoll

A simple method was developed for separation of cells from nasopharyngeal secretion for the diagnosis of respiratory virus infections by immunofluorescence microscopy. The diluted specimen, containing dithiothreitol to break up the mucus, was centrifuged once through a cushion of 20% Percoll (colloidal silica, a density gradient medium), which permitted sedimentation of cells through the cushion, but retained mucus on top of it. The pelleted cells were resuspended, and microscope slides were then prepared by standard techniques. The Percoll centrifugation method was also applicable for sputum and bronchoaveolar lavation specimens. Immunofluorescent antibody staining of nasopharyngeal secretions prepared by the described method was more sensitive than enzyme immunoassay for the detection of respiratory syncytial virus.

Gm allotypes influence the production of IgG3 but the effect is age-dependent

Serum concentrations of IgG3 were found to be higher in Gm-f-positive (= b-positive) than in f-negative individuals except in young children. Young children aged 3-4 months had a mean concentration of 0.24 g/l of IgG3 regardless of allotype. The concentration gradually rose with age in f-positive individuals to a geometric mean of 0.56 g/l in adults but it remained essentially unchanged in f-negative people. A corresponding allotype effect was seen in influenza-specific antibody responses. While the total IgG response (mainly IgG1) was equally strong in f-positive and in f-negative patients, f-positive (= b-positive) patients produced more IgG3 antibodies than f-negative patients. The difference between geometric mean values of opposite homozygotes (f/f versus f-negative) was 2.3-fold (p = 0.0113). This finding indicates that the b-positive gamma-3 allele is more productive than the g-positive allele.

Immune complex assays and stored normal human sera.

Abstract Normal human sera were tested for immune complexes (IC) by 3 different tests: Clq-binding ELISA (ClqB-ELISA), conglutinin binding ELISA (KgB-ELISA) and platelet iodinated protein A test (PIPA). 118 sera stored for various times were tested in 3 separate groups: 45 freshly obtained sera (group 1), 38 sera stored at -20°C for 1.5 years and frozen and thawed 5–8 times before testing (group 2), and 35 sera stored at -20°C for 4 years (group 3). Group 1 formed the ‘control’ group and test values exceeding the mean plus 3 S.D. of the controls were considered positive. By the ClqB-ELISA, 8% and 6% respectively of group 2 and 3 sera were positive. In the KgB-ELISA, the group 2 and 3 values were 37% and 29%, and in the PIPA test, 24% and 20%. The KgB-ELISA results correlated significantly ( r = 0.36, P P Special care in evaluating IC assay results should, therefore, be taken when stored sera are being tested for IC. Adequate controls of the same storage age should be included. High serum IgG concentrations may correlate with positive results in IC assays.

Sensitive interferon assay based on immunoenzymatic quantification of viral antigen synthesis.

A sensitive enzyme immunoassay (EIA) for determining the biological activity of human interferon was developed. Green monkey kidney (Vero) cells and human embryonic lung (HEL) cells were grown in microtitre plates, treated with leukocyte interferon (IFN alpha) and infected with vesicular stomatitis virus (VSV). Cells were fixed with paraformaldehyde and permeabilized with Triton X-100. Viral antigen synthesis was measured by labelling the cells with VSV antiserum followed sequentially by protein A horseradish peroxidase conjugate and o-phenylenediamine. Interferon activity was detected as a lowering of the absorbance value from that of the virus control wells, reflecting the inhibition of virus protein synthesis by interferon. The minimum amount of interferon producing statistically significant (P less than 0.01) decrease of absorbance in Vero cells was 1-5 international units (I.U.)/ml as in the standard plaque reduction test the detection limit was 7.5 I.U./ml or more. In HEL cells the detection limit was 1 I.U./ml measured by EIA. The EIA for interferon activity is at least as sensitive as the traditional plaque reduction test. It is reproducible, easy to automatise and requires 7-10 times less cell culture materials than the plaque reduction test. We find it preferential especially when large numbers of specimens with limited volumes are to be analysed for interferon activity.

C3b binding and IgG binding onto platelets in hepatitis B virus infection

Sera from patients with acute hepatitis B, hepatitis B surface antigen (HBsAg)-positive chronic hepatitis, and from asymptomatic HBsAg carriers were evaluated by enzyme-linked immunosorbent assay and by platelet 125I-labeled staphylococcal protein A test for the presence of C3b-binding IgG, IgM, and IgA activity (immunoconglutinins and circulating immune complexes of IgG, IgM, and IgA classes), and for the presence of IgG binding onto platelets, respectively. Significantly elevated C3b-binding IgG, IgM, and IgA activity (P < 0.001) was found in patients with acute hepatitis B, C3b-binding IgG activity (P < 0.01) in chronic hepatitis patients, but normal C3b-binding Ig activity in HBsAg carriers and controls. Similarly, platelet-bound IgG was significantly elevated in patients with acute hepatitis B and HBsAg-positive chronic hepatitis (P < 0.001). Serial studies in acute hepatitis B demonstrated that C3b-binding IgG and IgA activity significantly correlated with serum aspartate transaminase and bilirubin (P < 0.01) and platelet-bound IgG with aspartate transaminase values (P < 0.05). In addition, a significant association of C3b-binding IgG activity with platelet-bound IgG was found (P < 0.01). The results suggest that C3b-binding ELISA and PIPA tests may have clinical and immunopathogenic importance in studies on hepatitis B infection.