Single radial complement fixation test for assaying antibody to influenza virus type-specific antigens

An immunodiffusion technique in agarose is described for assay of complement-fixing antibodies against the type-specific soluble antigen of influenza virus. Under the test conditions, positive human serum produced a definite unlysed zone around the well, and the annulus area showed a high correlation with the antibody level in a conventional complement fixation test with log2 serum titer. This paper also describes the use of this method as a diagnostic procedure for the assay of antibodies against soluble antigens of influenza A and B viruses in paired human sera collected from persons infected with the virus or who received ether-split vaccine. This method appears to more sensitive and gives more consistent results for serodiagnosis of infection cases than do the hemagglutination inhibition, neuraminidase inhibition, and complement fixation tests. Our results suggest that the single radial complement fixation test can provide a simple and reliable method for serodiagnosis of influenza virus infection.     

Hemagglutinin-specific enzyme-linked immunosorbent assay for antibodies to influenza A and B viruses.

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies present in human serum or nasal washes directed against influenza A or B hemagglutinin glycoproteins. The assay was modified to measure the immunoglobulin isotype specificity of the anti-hemagglutinin response in serum and nasal secretions. In the postinfection sera anti-hemagglutinin of the immunoglobulin G isotype was predominant, whereas in nasal secretions the antibody was predominantly immunoglobulin A. The antibody response detected by the ELISA manifested hemagglutinin subgroup specificity. In addition, there was a good correlation between the ELISA antibody titer and the hemagglutination-inhibition or neutralizing antibody titer. The ELISA was more sensitive than the hemagglutination-inhibition assay, and the range of antibody titers measurable by ELISA in human serum was from less than 1:20 for children who had never experienced influenza infection to 1:400,000 for adults convalescing from a secondary infection. With more sensitive tests to detect antibody to the influenza hemagglutinin it should be possible to determine the relative contribution of local and systemic immunity to resistance to influenza virus infection.     

Measurement of antibody to influenza virus neuraminidase by single radial hemolysis in agarose gels.

A simple method of assaying anti-influenza neuraminidase antibodies in human sera was described. Suitable antigenic hybrid viruses were adsorbed to sheep erythrocytes, which were then incorporated into agarose gels. When sera were introduced into wells cut in the gels, zones of hemolysis were observed in the neighborhood of those containing neuraminidase antibodies. There was a direct relationship between zone size and antibody titer. No purification of adsorbed viruses was necessary. The test was rapid, required very simple reagents, gave results that agreed well with those given by conventional techniques, and appeared to be the most sensitive of four methods evaluated. Studies of cross-reactions by hyperimmune sera against homologous and heterologous neuraminidases and of absorption of neuraminidase antibodies from human sera indicated a high degree of specificity. The technique seems to be suitable for large-scale epidemiological investigations.     

Neurotropic influenza type A viruses

Influenza A/turkey/England/63 is neurotropic for mice. Substitution of the hemagglutinin gene of this virus by the corresponding gene of A/FPV/ Rostok /34 virus results in the loss of the neurotropic properties of the original virus. Examination of recombinants produced by hybridization of parental strains nonpathogenic (or weakly pathogenic) for newborn mice revealed recombinants highly virulent for this host. A correlation between constellation of genes and neurovirulence for mice was established. After intranasal administration neurovirulent viruses were shown to be able to penetrate into the brain of the infected animal along the trigeminal nerve escaping the blood stream.     

Use of single radial haemolysis for assessing antibody response to influenza virus vaccines in animals

The value of the single radial haemolysis (SRH) test as a possible replacement for the haemagglutination-inhibition (HAI) test for the estimation of antibodies against influenza was assessed in three animal models. The serum antibody response was measured by both assay systems; correlation of the two tests was assessed using regression analysis. The study showed that when the response to a single immunisation was determined, the ferret model gave satisfactory correlation of SRH and HAI, whilst in the mouse and hamster models poor correlation was observed. Correlation was only improved in the mouse model when an immunisation schedule that mimicked the human situation of a background exposure to different strains of influenza virus was used. Since influenza vaccine efficacy is usually assessed in animals using a single immunisation we suggest that the SRH is not acceptable for use in either hamsters or mice, but is acceptable where the ferret model is involved.     

A simple screening assay for receptor switching of avian influenza viruses.

BACKGROUND: Adaptation of the receptor-binding preference from alpha2,3- to alpha2,6-linked sialic acid is an essential step for an avian influenza virus to transmit efficiently in human population and become a pandemic virus. The currently available assays for receptor-binding preference are complex and not widely available. OBJECTIVES: A simple high-throughput screening assay will facilitate early detection of a potential pandemic virus, which is crucial for the prevention and control of the possible pandemic. We wanted to develop a simple assay to differentiate influenza viruses with alpha2,3- or alpha2,6-linked receptor-binding preference. STUDY DESIGN: The assay employs a specific sialidase (from Salmonella thyphimurium) that can eliminate alpha2,3-linked sialic acid from red blood cells. A reduction of hemagglutination titer indicates alpha2,3-linked receptor preference in this assay. RESULTS: Using a panel of H5N1 avian influenza isolates and H1/H3 human influenza isolates, as well as mutated H5 reverse genetics virus, the assay could accurately differentiate the viruses according to their receptor-binding preference. Furthermore, the assay was sufficiently sensitive to detect a minor variant with alpha2,6-linkage-specificity in a background of alpha2,3-linkage-specific virus. CONCLUSIONS: We have developed a simple screening assay capable of detecting avian influenza viruses that have switched their receptor-binding preference.     

A re-examination of the single radial haemolysis technique for the assay of influenza anti-neuraminidase antibodies in human sera

Summary A new study is described of the use of single radial haemolysis (SRH) for the measurement of antibodies to influenza virus neuraminidase (NA). The technique is known to be consistently successful in the assay of anti-haemagglutinin (HA) antibodies, subject only to the condition that the indicator virus belongs to an appropriate serotype. Its adaptation to the measurement of anti-NA is, however, more difficult. The virus used must be a recombinant which contains a specific NA and an irrelevant HA. However the present experiments showed that the two recombinants MRC-3 and X-38, which contain the same NA but a different HA, gave different results. Other properties of recombinants, including rates of attachment to and elution from red cells, many affect the results. The chemical NA-inhibition test (NI), although requiring the use of antigenic hybrids, did not produce these discrepancies. However it appears possible to exploit the simplicity and convenience of SRH for mass survey of anti-NA, if individual hybrid recombinants can first be shown to yield results comparable to those obtained by NI.With 2 Figures     

Single radial complement fixation test using complement film. Assay of the antibody response to strain and type specific antigens of influenza virus

A stabilized modification of the single radial complement fixation test in gel (SRCF) was developed for detecting influenza antibodies. The principle of the test is the use of a single-step procedure with the following reagents: (1) Agarose plate containing influenza antigen and antibody coated erythrocytes (EA). (2) Thin plastic film coated with dried complement. By filling the wells cut in the agar with the heat inactivated serum samples and covering the agar surface with the complement film, a zone of unlysed cells surrounded by a haemolytic area appears after overnight incubation for 16–18 h at 4°C and 1–2 h at 37°C.The squares of the zone diameter were measured for estimating the antibody quantity by using CF(S) and virion antigen of influenza virus, and the type-specific antibody was demonstrated by using CF(S) antigen, while the strain-specific antibody was demonstrated by using virion antigen. An excellent correlation was demonstrated for antibody titres between conventional CF and SRCF with CF(S) antigen, on the one hand, and, between conventional HI and SRCF with virion antigen, on the other.The simplicity of the procedure and reproducibility of the results might prove the new test tobe the most useful method for routine serodiagnosis and serological survey of influenza virus infections.     

Single radial hemolysis test for quantitation of complement-fixing antibodies to non-hemagglutinating viruses.

We describe two enzyme-linked immunosorbent assays for rotavirus antigen in feces, which were designed to be as sensitive and specific as possible, and easy to use anywhere. Both are indirect methods, using the antibody capture method, but the second assay utilizes a rotavirus group-specific monoclonal "detecting" antibody instead of the hyperimmune polyvalent guinea pig antisera used in the first assay. Both tests were found to be more sensitive than electron microscopy for detecting virus. To develop these tests, solid phase, antiserum production methods, treatment of the test antigen with EDTA, substrate, stability of reagents, and the need for confirmatory "blocking" tests were all examined. The first assay described is that used at present by the World Health Organization for their worldwide diarrheal disease control program.     

Detection of cells producing surface antigen-specific antibody to influenza viruses.

B cells producing antibodies to influenza virus antigens were detected and quantitated by a hemolytic plaque assay. Responses of mice after primary infection and immunization with influenza viruses were measured and compared with responses after secondary immunization. The B-cell responses were specific and differentiated between A and B influenza viruses and between different subtypes of A influenza viruses. Responses to closely related influenza A virus strains of the H3N2 subtype cross-reacted but could also be differentiated. Cells secreting antibody to either of the virus surface antigens (hemagglutinin and neuraminidase) could be separately enumerated. Evidence that immunoglobulin G- secreting cells are detected in the assay without the use of facilitating anti-immunoglobulin G sera is presented.     

A single radial immunodiffusion test for antibodies to Newcastle disease virus

A single radial immunodiffusion (SRD) test for the measurement of antibodies to Newcastle disease virus (NDV) is described. The test involved incorporation of concentrated intact NDV into a gel of agarose with low setting temperature and measurement of opalescent zones produced by the diffusion of antibodies to the NDV surface antigens from wells cut in the gels. With both fowl and turkey antisera the sizes of the opalescent zones showed strong correlation with the haemagglutination inhibition (HI) titres of the sera. The possible uses of the SRD test in epizootiological studies are discussed.     

Detection of antibody to immunodeficiency viruses by dot immunobinding assay.

The dot immunobinding assay (DIA), a modified enzyme immunoassay (EIA), has been demonstrated to be a highly sensitive and specific assay for the detection of antibody to a number of viruses. Different laboratory procedures are available for detecting antibody to the immunodeficiency viruses; however, these procedures require a certain amount of sophisticated equipment and trained personnel. Further, commercial kits for detecting antibody to human immunodeficiency virus, as now available, are not easy to use in the nonlaboratory setting. The DIA, as described herein, may be formatted to test up to 30 serum samples and is designed to be used in the absence of laboratory equipment. To determine the effectiveness of the DIA as a test kit for the detection of HIV and human T-cell leukemia virus type I (HTLV-I) antibodies, the kit was compared with commercial EIA and Western blot (WB; immunoblot) kits. Testing approximately 1,000 human serum samples for HIV antibody by DIA and EIA revealed a total agreement of 98.1%, a specificity of 99.0%, and a sensitivity of 95.9%. For 804 serum samples tested (200 were tested independently in two laboratories), eight results were discrepant: four DIA negatives which were EIA borderline positive and four DIA positives which were EIA negative. Testing the eight discrepant sera by immunofluorescence assay and WB resulted in their being either negative or indeterminate. The four DIA positives were indeterminate by WB. Close agreement was obtained when the remaining sera were compared by DIA, EIA, and WB. Of interest was finding that the DIA results compared favorably with those obtained by WB. Twenty-six suspect HTLV-I-positive serum samples tested by DIA also gave results comparable to those obtained by EIA and WB.     

Application of the single radial complement fixation test for serodiagnosis of influenza, respiratory syncytial, mumps, adeno type 3, and herpes simplex type 1 virus infections

A stabilized modification of the single radial complement fixation (SRCF) test in gel was developed for detecting various virus antibodies. The principle of the test is based on the use of a single-stage procedure with an agarose plate containing virus antigens and antibody-coated erythrocytes, and thin plastic film coated with complement. By filling the wells in the agar plate with a 1:4 diluted heat-inactivated sera and covering the agar surface with a complement film, a zone of unlysed cells surrounded by a hemolytic area appears after incubation overnight at 4 degrees C and then for 1-2 h at 37 degrees C, depending on the antibody titers. The SRCF antibody titer is calculated numerically from the square of the diameter of the unlysed cell zone. The stability of reagents could be significantly improved using thin complement film and several stabilizers. When this test was used for serodiagnosis of influenza, respiratory syncytial (RS), mumps, adeno virus type 3 and herpes simplex type 1 virus infections (using a total of 400 sera), excellent correlations were demonstrated for antibody titers between conventional complement fixation (CF) and SRCF titers. Furthermore, the expression of antibody titer as an SRCF unit with consecutive value, produced results sensitive to fluctuations in the antibody titers. The simplicity of the procedure, stability of the reagents, and excellent correlation with the conventional CF test might make this a useful test for routine serodiagnosis and seroepidemiological survey of various virus infections.     

A simple plaque assay for influenza A2 viruses and its application to antiviral screening

Summary Influenza A₂/Singapore and A₂/Hong Kong virus strains formed plaques in monolayers of monkey kidney and dog kidney cells. Using a serum-free overlay containing Earle's balanced salt solution with 1% lactalbumin hydrolysate, 1.4 g/1 sodium bicarbonate and 0.6% agarose, plaque formation occurred in 4–7 days. By incorporating compound in the overlay, plaque inhibition was demonstrated by Amantadine, Rimantadine and the isoquinoline derivatives designated Pfizer UK-2054, UK-2617, UK-2762 and UK-2888.     

The single radial immunodiffusion assay highlights small antigenic differences among influenza virus hemagglutinins

The results of single radial immunodiffusion assays of influenza virus hemagglutinin were found to be greatly altered by small antigenic differences between test and reference strains. When such differences were present, the precise specificity of the antiserum used had a critical effect on the measured hemagglutinin antigen content obtained.     

Accuracy of single radial hemolysis test for rubella immunity when internal reference standards are used to estimate antibody levels.

The accuracy and reproducibility of antibody levels obtained by single radial hemolysis with six internal reference sera were evaluated. The test was performed in a clinical laboratory for routine assessment of immunity to rubella infection over a period of 1 year. A linear relationship exists between the antibody titer (expressed in log dilution) and zone diameters. In 43 of 44 test runs the correlation coefficient of the standard curve was over 0.990. Prediction limits of 95% around the curve showed that on replication of the test, zone diameters could be found within less than half a doubling-dilution step. The antibody level can thus be determined more accurately by the single radial hemolysis test than in the conventional hemagglutination inhibition test. This is particularly important in assessing immunity when antibody titers are low, since the hemagglutination inhibition test is less reliable then. The use of standard sera calibrated international units would render results of different laboratories comparable and allow standardization at threshold values.     

Immunoglobulin-specific radioimmunoprecipitation assays for quantitation of nasal secretory antibodies to hemagglutinin of type A influenza viruses.

Radioimmunoprecipitation (RIP) assays were developed to selectively quantitate class-specific antibodies to purified hemagglutinins (HA) of type A influenza virus in nasal secretions. Rabbit anti-human secretory piece of immunoglobulin A (IgA) and rabbit anti-human IgG were used as second antibodies. A third antibody, goat anti-rabbit IgG, was incorporated into the system to separate immune complexes formed between iodinated HA, nasal wash test specimen, and second antibody. The utilization of this reagent avoided the need for large quantities of IgA and IgG antibody-negative carrier secretions. Nasal was specimens obtained from 14 adults immunized with an inactivated type A influenza virus vaccine were evaluated by RIP and viral neutralization assays. Significant homologous postvaccination secretory IgA and IgG antibody levels were demonstrable in 13 (93%) of individuals by RIP, whereas only 5 (36%) exhibited rises by viral neutralization tests. Moreover, the geometric mean IgA and IgG antibody levels were at least 20- and 37-fold greater than the neutralizing antibody titer. The pattern of heterologous immunoglobulin-specific antibody responses tended to be similar to those observed with the homologous HA subunit.     

Antibodies to type A influenza viruses in sera from nonhuman primates

Summary The results of serological testing of nonhuman primate sera obtained over a four-year period showed a high incidence of antibodies to influenza virus strains of the H2 and H3 hemagglutinin sub-types. This would indicate that outbreaks of type A influenza virus infection occurred in certain primate species and suggest another possible reservoir for influenza virus in nature. Hemagglutination-inhibition (HI) testing of sera collected from African green monkeys captured in the Kenya-Tanzania area of East Africa demonstrated significant antibody titers to A/Hong Kong (HK)/68 (H3N2) virus in serum samples obtained 8 to 10 months prior to the first report of influenza-like illness in East Africa, and 3 to 5 months prior to the first report of outbreaks of acute respiratory disease in southeastern China due to A/HK/68 (H3N2). The results suggest that certain species of nonhuman primates may be involved in the epidemiology of influenza due to their close association with human living areas.