Evaluation of the Single Radial Hemolysis Test for Measuring Hemagglutinin- and Neuraminidase-Specific Antibodies to H3N2 Influenza Strains and Antibodies to Influenza B

Antibodies to the H3 hemagglutinin of influenza A virus could be specifically measured by single radial hemolysis (SRH) when test antigens were recombinant viruses containing the relevant H3 hemagglutinin antigen and irrelevant Neq1 neuraminidase of A/equine/Prague/1/56 virus. Antibodies to influenza B virus could also be measured by the SRH technique. Antibody rises to influenza A or B virus measured by SRH agreed with results of hemagglutination inhibition (HI) tests for about 80% of the sera tested, including sera from volunteers receiving killed influenza vaccine and sera from patients naturally infected with influenza. Correlation between antibody titers measured by SRH and HI was also good. Antibodies to the N2 neuraminidase of influenza A virus could be specifically measured by SRH when test antigens were recombinant viruses containing the relevant N2 neuraminidase antigen and irrelevant Heq1 hemagglutinin of A/equine/Prague/1/56 virus. The SRH test for neuraminidase antibodies was more strain specific than was the SRH test for hemagglutinin antibodies. Probably for this reason, agreement between neuraminidase antibody determinations in human sera by the SRH test and by the neuraminidase inhibition test was poorer than agreement between the SRH test for hemagglutinin antibodies and the HI test.     

Clinical reactivity and immunogenicity of hemagglutinin influenza vaccine

Summary Subjects aged 3–6, 16–17 and 27–50 years were vaccinated with one dose of hemagglutinin influenza virus vaccine. Clinical reactions, hemagglutination-inhibiting (HI) and strain- and type-specific complement-fixing (CF-V and CF-S) antibodies were determined in sera taken before and four weeks after vaccine administration. The results indicated that the reactogenicity of the vaccine was very low. The HI antibody response differed with the age of the vaccinees, apparently being conditioned by prior exposure to the various influenza virus subtypes. The results of CF tests using strain-specific V antigens corresponded in general with HI tests, with two marked exceptions. In the youngest group nearly half of the subjects developed CF antibody to V-Swine, while all of them remained without antibody detectable in the HI test. However, when V antigen was used instead of intact virus as hemagglutinin, the post-vaccination sera of these subjects also reacted positively in the HI test. Secondly, a number of prevaccination sera from persons aged 27–50 years possessed CF antibody to A/PR 8 in the absence of homologous HI antibody. Among these subjects the antibody response to both A/PR 8 and Swine was more marked in the CF test than in the HI test. After vaccine administration most of the subjects developed antibody or responded by an antibody increase to the S antigens of both influenza A and B. No significant differences were found after intradermal (0.1 ml) and subcutaneous (0.5 ml) administration of one dose of vaccine.     

Assessment of the sensitivity of immunoprecipitation for the serodiagnosis of influenzal-bacterial pneumonias]

A comparative examination of 226 paired sera from patients with pneumonia was carried out by CFT, HI, neuraminidase activity inhibition (NI) and double immunodiffusion. A correlation of the results of agar gel precipitation and the CFT and HI tests was observed. Convalescent sera contained antibody to influenza virus ribonucleoprotein frequently, less so to its neuraminidase or hemagglutinin. The precipitation test was shown to be highly sensitive, easy to perform, and therefore should be used in examinations of sera from patients with influenza-bacterial pneumonia.     

Detection of anti-H5 responses in human sera by HI using horse erythrocytes following MF59-adjuvanted influenza A/Duck/Singapore/97 vaccine

Haemagglutination-inhibition (HI) tests are a simple method used to assess immune responses to influenza haemagglutinin. However, HI tests are insensitive at detection of antibody responses to avian haemagglutinin after vaccination or natural infection, even in the presence of high titres of neutralising antibody or virus isolation. Avian influenza viruses preferentially bind to sialic acid receptors that contain N-acetylneuraminic acid alpha2,3-galactose (alpha2,3Gal) linkages while human viruses preferentially bind to those containing N-acetylneuraminic acid alpha2,6-galactose (alpha2,6Gal) linkages. By using horse erythrocytes in the HI test and thereby increasing the proportion of alpha2,3Gal linkages available for binding, we are able to demonstrate improved detection of antibody to avian H5 in human sera following vaccination with MF59-adjuvanted A/Duck/Singapore/97 surface antigen vaccine. This modified HI test was more sensitive in detection of anti-H5 antibody evoked by revaccination of primed subjects and may be useful in assessing potential avian HA vaccine candidates.     

Simultaneous determination of the level of antibodies to influenza virus surface and internal proteins by enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay (ELISA) has been adopted for simultaneous determination of the levels of antibodies to different influenza virus proteins in human sera with known haemagglutination-inhibition (HI) titre. Whole virus of serotypes H1N1 and H3N2, haemagglutinin (HA), matrix (M) and nucleoprotein (NP) proteins have been used as antigens. For detection of antibodies bound to the antigen, peroxidase labelled Staphylococcus protein A conjugate has been used. Correlation of the ELISA and HI titres of anti-HA antibody has been demonstrated. The use of isolated HA as antigen increased the specificity of ELISA. The analysis of human reconvalescent sera has shown that increase in the titre of antibodies to internal proteins does not always coincide with the increase of antibody level to HA. Out of 8 sera with significant increase of the HI titre to the H3 subtype 5 specimens showed 4-fold increase of antibody titre to NP protein. The antibody titre to M protein was elevated in 2 sera only, while 1 serum showed no rise of antibody response to the tested viral proteins.     

Levels of immunoglobulins and antibodies to haemaglutinin and neuraminidase of influenza virus in nasal secretions after natural infection.

Nasal washings (NW) from 16 influenza patients in the course of an epidemic in November and December, 1974 were examined for the presence of influenza virus, immunoglobulins (Ig) and titres of haemagglutination inhibiting (HI) and neuraminidase inhibiting (NI) antibodies. Influenza virus identical with A/Port Chalmers/1/73 (H3N2), increased levels of IgA and occasionally IgG, and specific antibodies were detected in the NW. The dynamics of HI and NI antibody formation did not differ substantially, but there were individual differences in titres and persistence of antibodies. Convalescent sera always contained increased levels of HI and NI antibodies. In some cases, the titres of antibody to viral ribonucleoprotein did not increase.     

Antibody reactive in antibody-dependent cell-mediated cytotoxicity following influenza virus vaccination

The antibody reactive in antibody-dependent, cell-mediated cytotoxicity (ADCC) to influenza virus-infected cells was measured in two groups of seven volunteers each, before and after immunization with inactivated or live attenuated A/Victoria/3/75 influenza virus vaccines. Age-matched controls were seven adult individuals who experienced natural influenza infection due to A/Victoria/3/75-like virus strain. After inactivated whole influenza virus immunization all the subjects showed a significant rise of the antibody reactive in ADCC (from a mean value of 4.7% to 17.1% cytotoxicity, before and 5 weeks after immunization, respectively) as well as of hemagglutination inhibition (HI) antibody (fourfold or greater increase). These immune responses were similar to those observed among naturally infected controls. After live attenuated virus vaccination, no significant increase in titer of antibody reactive in ADCC was detected, even though the vaccine induced significant increase of HI antibody titer. Little correlation was found between ADCC and HI antibody rises in sera of recipients of inactivated virus vaccine and of naturally infected individuals, while, in live attenuated influenza virus vaccinees, the rise of HI antibody titer did not correspond to a significant increase of ADCC antibody titer; several subjects who developed a significant rise in ADCC antibody titer did not show significant variation in antibody to neuraminidase and/or to complement fixation influenza virus antigens.     

Antibody response in humans to influenza virus type B host-cell-derived variants after vaccination with standard (egg-derived) vaccine or natural infection.

Hemagglutination inhibition (HI) and neutralization tests were used to determine antibody responses to egg-derived and Madin-Darby canine kidney (MDCK)-derived influenza B virus (B/England/222/82) in paired sera from persons naturally infected with influenza B and in persons vaccinated with standard egg-derived inactivated influenza vaccine. When tested by HI, the MDCK-derived antigen gave significantly higher (8- to 12-fold) geometric mean titers (GMT) in convalescent-phase sera from persons naturally infected during community outbreaks, as well as more 4-fold titer rises, than did tests with egg-derived antigen. When tested by neutralization, however, the convalescent-phase sera GMTs were only threefold higher with the MDCK-derived antigen and an equivalent number of fourfold titer rises were detected with both antigens. With postvaccine sera, the MDCK-derived antigen gave GMTs that were threefold higher than those obtained with egg-derived antigen in both the HI and neutralization tests and both antigens detected an equivalent number of fourfold titer rises in HI and neutralization tests. Sucrose gradient-fractionated egg-derived antigen showed a single peak of hemagglutinin activity corresponding to whole virions, whereas MDCK-derived antigen contained two distinct peaks of hemagglutinin activity, one of which had a lower sedimentation rate. The overall findings indicate that the egg-derived antigen in the vaccine induced HI and neutralizing antibody to both egg- and MDCK-derived variants and suggest that titers of antibody to MDCK-derived virus may be affected by the physical form of the hemagglutinin antigen.     

Detection of antibodies to influenza virus M protein by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay test system was developed in which purified influenza virus M protein was used for the detection of M antibody in human sera. Antibody levels to influenza A virus M protein were monitored in sera from a vaccine study population by using an enzyme-linked immunosorbent assay technique with purified M protein as the adsorbent antigen. A 10-fold variation in titers of preexisting M antibody was observed in this population of young adults. Increases of anti-M titer of 7- to 24-fold were observed upon immunization with Formalin-inactivated vaccine or after natural infection. The antibody response to M protein was dissociated from the response to the hemagglutinin or neuraminidase antigens. The M antibody response preceded or was coincident with the antibody response to H1 hemagglutinin upon natural exposure to circulating virus.     

Bile immunoglobulin of the duck (Anas platyrhynchos). II. Antibody response in influenza A virus infections

The capacity of the IgM-like bile immunoglobulin (IgX) of the duck (Anas platyrhynchos) to express antibody activity to H3N2 influenza A viruses, and the dependence of this activity on the co-existence of serum IgM antibodies were investigated. Ducklings infected orally and intranasally at 15-29 days of age with viruses isolated from different host species were examined for haemagglutination-inhibiting (HI) antibodies in biles and sera 16-29 days after infection (p.i.). All biles had antibodies associated with IgX; all sera had antibodies associated only with the 7.8S IgG. Following oral infection of birds 42-days-old with influenza A/duck/HK/7/75 virus, serum HI antibodies were an initial IgM response occurring from 5-12 days p.i., followed by the appearance of 7.8S IgG antibodies. Virus-neutralizing (VN) antibodies in serum were also biphasic; isotype classification was not attempted. Bile IgX developed HI and VN activity. HI antibodies reached peak titres 12 days p.i. and fell to low levels by 24 days p.i. VN antibodies also reached peak titres 12 days p.i., but thereafter persisted at quite high levels throughout the experiment. Development of high titres of antibody in bile coincided with the termination of virus excretion in faeces. These experiments confirm that bile IgX of the duck can function as antibody in response to influenza A viruses, and that its activity appears to be independent of serum IgM. Its possible relevance in determining survival of virus in the intestine is discussed.     

Improved rubella hemagglutination inhibtion test: inactivation of non-immunoglobulin hemagglutination inhibitors by phospholipase C.

A method using phospholipase C (PL-C) for removing nonspecific inhibitors (NSI) of rubella virus hemagglutinin is described. PL-C was found to hydrolyze NSI without altering the hemagglutination inhibition (HI) activity of the specific antibody and could be used to remove NSI in the rubella HI test by using formalinized erythrocytes, which resisted the enzymatic action; fresh erythrocytes were lysed by PL-C. The HI test using PL-C treated sera gave true measurements of actual rubella antibody content, and HI titers of PL-C treated sera were identical or equivalent (+/-1 dilution) to those of sera treated with dextran sulfate and CaCl2 (DS-C). Thus, the PL-C method gave results as reproducible and reliable as the DS-C method and was more convenient.     

深圳地区人和鸡群中H9N2亚型流感病毒病原学和血清流行病学调查

目的：了解甲型流感病毒N9N2亚型毒株在深圳地区鸡群和人群中的分布。方法：采用常规的鸡胚双腔法来分离病毒。抗体测定，采用红细胞凝集抑制（HI）试验和中和试验测定法。结果：从深圳地区农贸市场鸡群中分离到27株H9N2亚型流感病毒，但未能从人群中分离到H9N2病毒。约有26％人血清中检测到H9亚型毒株的抗体，（HI滴度≥20），同时还发现抗体阳性率和几何均数随人群年龄增长而增高，同时与职业有关。然而，在鸡群中H9毒株的抗体阳性率仅为7％。结论：禽H9N2毒株不仅能感染人，而且在深圳地区人群和禽类中较为广泛的分布。人H9N2很大可能来源于鸡的H9N2毒株。     

New passive hemagglutination assay kit that uses hemagglutinin-sensitized erythrocytes for detection of rubella antibodies.

A new passive hemagglutination assay for the detection of antibodies to rubella virus hemagglutinin (PHAST-Rubella) was compared with the hemagglutination inhibition (HI) test and another passive hemagglutination test that uses a soluble rubella virus antigen (SA-PHA). When the immune responses of vaccinated individuals were monitored, similar rises in antibody titer were detected by HI or PHAST-Rubella, whereas the rise in titer detected by SA-PHA was delayed. Early-phase vaccine-induced immunoglobulin M antibody analyzed by sucrose gradient fractionation was detected to the same degree by HI and PHAST-Rubella, but early-phase immunoglobulin G antibody reacted more strongly in the HI test. When acute and convalescent serum pairs from rubella-infected individuals were evaluated, a fourfold rise in titer was detected by PHAST-Rubella and HI in 15 of 15 pairs, whereas SA-PHA, which is not intended for detecting antibody titer rises in acute infections, detected a rise in titer in only 3 of 15 pairs. In studies to determine rubella immune status, testing of 1,078 premarital and random serum specimens resulted in 98.6% agreement among the three methods in identifying rubella antibody-positive and -negative individuals. For the quantitative PHAST-Rubella procedure, a coefficient of correlation of 0.93 was obtained, in comparison with HI, when a panel of 40 characterized sera were tested. These results indicate that PHAST-Rubella reagents can detect rubella antibodies as well as HI reagents and thus may be used as a fast and accurate means of determining rubella immune status and for the quantitation of rubella antibodies.     

Sialic acid receptor specificity on erythrocytes affects detection of antibody to avian influenza haemagglutinin

Haemagglutination-inhibition tests (HI) are used to detect increases in influenza antibody in serum. However, they are relatively insensitive for the detection of human antibody responses to avian haemagglutinin, even in the presence of high titres of neutralising antibody after confirmed infection or vaccination. Human influenza viruses bind preferentially sialic acid containing N-acetylneuraminic acid alpha2,6-galactose (SAalpha2,6Gal) linkages while avian and equine viruses bind preferentially those containing N-acetylneuraminic acid alpha2,3-galactose (SAalpha2,3Gal) linkages. Increasing the proportion of SAalpha2,3Gal linkages on the erythrocytes used, by enzymatic modification or change of species, improves the ability of erythrocytes to bind to avian influenza strains and thereby improves the sensitivity of detection of antibody to avian and equine HA in a range of mammalian and human sera using HI tests.     

Nonspecific inhibitors of coronavirus OC43 haemagglutination in human sera

Antibodies against the human coronavirus OC43 in human sera were measured by haemagglutination inhibition (HI), complement fixation (CF), and radial diffusion-haemolysis in gel (HIG) techniques. The apparent HI titres in a fraction of sera with no antibodies detectable by the two other methods were found to be reduced considerably after treating the sera with phospholipase C (PLC). The PLC treatment also reduced the apparent HI titres in some sera containing variable amounts of CF and/or HIG antibodies, but did not affect antibody determinations by the two latter methods.These results suggest that false positive results can be obtained in the OC43 HI test unless the sera are treated with phospholipase C before the assay.     

An enzyme linked immunoassay for estimation of antibodies to Newcastle disease virus strains

The sensitivity of enzyme linked immunoassay (ELISA) was compared to that of haemagglutination inhibition (HI) test in detection of humoral antibodies in birds vaccinated with Newcastle disease virus (NDV). The highest antibody titre at day 21 post-vaccination as detected by ELISA in the sera of vaccinated birds was 2560 by all strains in contrast to the HI titres of 640 to 1280. The antibody titre was higher in all pre- and post-vaccination sera as determined by ELISA than detected by HI test.     

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.     

Serological diagnosis of parainfluenza virus infections: verification of the sensitivity and specificity of the haemagglutination-inhibition (HI), complement-fixation (CF), immunofluorescence (IFA) tests and enzyme immunoassay (ELISA).

Using four serological tests paired sera were examined of 117 patients with acute respiratory diseases, in whom parainfluenza viruses (PIV) infection was demonstrated by virus isolation, and of 41 patients with typical clinical mumps symptoms. Comparative analysis showed the high sensitivity of IFA and ELISA. A significant rise of antibodies in convalescent sera with homologous antigen of PIV was found in nearly 100 percent of cases. Only the sera of youngest children with high titres of persisting maternal antibodies remained without seroconversion. Cross heterologous antibody responses could be found by means of ELISA in 45% and by IFA in 10%, of patients who in the past experienced infection with one or more PIV or mumps virus--apart from homologous antibody reaction. HI and CF test proved to be less sensitive for detection of postinfections antibodies, especially in primoinfections with PIV types 1 and 2.     

Serological evidence of viral orMycoplasma pneumoniae infection in acute maxillary sinusitis

Evidence for the involvement of viruses,Mycoplasma pneumoniae, andChlamydia spp. was studied by the complement fixation test in paired sera from 310 young adults (297 men and 13 women) with acute maxillary sinusitis. The diagnosis of acute sinusitis was confirmed by radiography and sinus puncture. Elevated antibody titres were found in 102 patients (33%). A four fold or greater titre rise was detected in 21.5%, and a high stable titre suggestive of recent viral infection was present in a further 11.5%. Adenovirus, influenza A and B viruses, andMycoplasma pneumoniae accounted for most of the elevated antibody titres. Elevated titres were found in 79 (32%) of the 245 patients with purulent maxillary sinusitis (pathogenic bacteria isolated in sinus secretion) and in 23 (35%) of the 65 patients with non-purulent sinusitis (no pathogenic bacteria isolated). About 90% of the fourfold or greater titre rises in bacteriologically negative cases were due to adeno- or influenza viruses. A fourfold rise in antibody titre was also found in 7 of 101 control patients (7%). The results of this study suggest that respiratory viruses andMycoplasma pneumoniae may be potential etiological agents in acute maxillary sinusitis, either alone or in combination with the common bacterial pathogens of sinusitis.     

2-site competitive immunoenzyme analysis based on monoclonal antibodies for the detection and titration of antihemagglutinating measles antibodies]

Using monoclonal antibodies (MCA) to measles virus (strain Leningrad-16) hemagglutinin, a competitive enzyme immunoassay (cEIA) was developed for the detection and titration of antihemagglutinating antibodies in human sera. Serum samples from children, collected in measles foci, were tested in cEIA, SP-EIA, HI, and PHA tests. The results evidence a high correlation of antihemagglutinin titres in cEIA and HI tests, the correlation coefficient of cEIA and PHA being 97.7%. The cEIA based on MCA to measles virus hemagglutinin is highly specific for the detection and titration of measles antihemagglutinins, it does not involve the use of simian erythrocytes. A significant advantage of this test system consists in the possibility of using unpurified antigen prepared from the infected cell lysate.