Vol. 5, No. 3

JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1977, p. 353-360 Copyright ©D 1977 American Society for Microbiology

Printed in U.S.A.

Evaluation of the Single Radial Hemolysis Test for Measuring Hemagglutinin- and Neuraminidase-Specific Antibodies to H3N2 Influenza Strains and Antibodies to Influenza B KHOSROW FARROHI,' FAKHRISSADAT K. FARROHI,' GARY R. NOBLE, HAROLD S. KAYE, AND ALAN P. KENDAL* World Health Organization Collaborating Center for Influenza, Respiratory Virology Branch, Virology Division, Bureau of Laboratories, Center for Disease Control, Atlanta, Georgia 30333

Received for publication 8 November 1976

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 Neql 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 Heql 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.

Antibodies to influenza virus nucleoprotein, hemagglutinin, and neuraminidase antigens may be measured by standardized complement fixation (CF) (17), hemagglutination inhibition (HI) (13), and neuraminidase inhibition (NI) (1) tests, respectively. These procedures have, however, certain recognized disadvantages, including the relatively low sensitivity of the CF test, the potential interference by NA antibody and nonantibody serum inhibitors in the HI test, and the complexity of the NI test which limits the number of samples that can be assayed in a given time. Alternative procedures have therefore been suggested. These include: the single radial diffusion (SRD) test (14) which may measure antibodies to any of the nucleoprotein, HA, and NA viral antigens as well as the viral matrix protein antigen (11); and the HI (9) and enhanced hemagglutination inhibition (NHI) (7) tests, and a passive hemagglutination test (5), which I Visiting Scientists from Tehran University, Basic Medical Science, Department of Microbiology and Immunology, Tehran, Iran.

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have been used for measuring neuraminidase antibodies. Each of these newer tests has its problems. The single radial diffusion test requires large amounts of purified virus, and the NHI and passive hemagglutination techniques require specialized biological reagents which introduce the possibility for errors due to the variable performance of these reagents (4). Recently another procedure, single radial hemolysis (SRH), has been reported to successfully measure antibodies to influenza A hemagglutinins (11), without sera having to be treated to destroy nonspecific inhibitors. The test was reported to be sensitive and did not require large amounts of virus antigen. Initial results were obtained in a study with wild-type influenza A viruses used as antigens. It has since been reported, however, that neuraminidase antibodies can also react in the SRH procedure (2). In this report we describe the evaluation of the SRH test for its correlation with the HI test under circumstances where reaction of neuraminidase antibodies in the SRH test was elimi-

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nated by using antigenic recombinant viruses. Additionally, we examined the SRH test for its ability to measure influenza B antibodies, so that the potential value of the test could be extended to include serodiagnosis of infections with all viruses of the influenza virus genus. At the same time, we evaluated the SRH test for measurement of neuraminidase antibodies in human serum, with the specific objective of determining whether antigenic drift of human influenza A neuraminidase would affect the reliability of the test. We did this because the test had been reported to be insensitive to neuraminidase antibodies other than those highly specific for the virus strain used as test antigen (2). MATERIALS AND METHODS Viruses. Recombinant influenza A viruses used in this study were originally obtained from Robert G. Webster. Influenza B/Hong Kong/5/72 used was a high-growth-yielding strain (BX 1) originally supplied to us by Merrell-National Laboratories, Swiftwater, Pa. Wild-type influenza A viruses were from the reference collection of this laboratory. Inactivated virus antigens were prepared by adding 0.1% beta-propiolactone (BPL) to allantoic fluid containing 0.025 M disodium monohydrogen phosphate and then incubating the mixture at 32°C for 2 h. The fluid pH was adjusted to 7.3 with 7% sodium bicarbonate solution when necessary. Preparation of SRH plates. The procedure was based on that described by Russell et al. (13). Packed sheep erythrocytes were mixed with an equal volume of 5 x 10-4 M potassium periodate in phosphatebuffered saline (PBS), pH 7.2, and reacted at room temperature for 10 min with gentle mixing. Viruscontaining allantoic fluid was then added to the cells in such a proportion that each 0.2 ml of erythrocyteperiodate mixture was reacted with 1,000 hemagglutinating units of virus. After 15 in, during which the mixture was gently shaken, the treated cells were washed three times in PBS and made up to a final 50% suspension. For each plate, 0.1 ml of treated cells (influenza A viruses) was mixed at about 42°C with 2.8 ml of a 1% solution of Indubiose A37 (L'Industrie Biologique Francaise) in PBS and 0.1 ml of guinea pig serum. Gels were poured into immunodiffusion plates and allowed to set; wells with diameters of 2 mm were then punched. The gels were stored at 4°C until used, within 1 to 3 days of preparation. Preliminary tests established that similar results were obtained with active or BPL-inactivated virus. Since inactivated antigens are preferable for use in diagnostic laboratories, all subsequent tests were conducted with BPL-treated virus. As described in Results, clearer zones of hemolysis were found for influenza B virus when gels contained 60% of the erythrocyte concentration used with influenza A viruses. Performance of the SRH test. Preliminary studies demonstrated that with undiluted human or animal convalescent sera, 1-,ul volumes gave satisfactory zone areas in most SRH tests. Larger volumes

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produced zone areas so extensive that they seriously limited the number of sera that might be tested on a single plate. Therefore, in all subsequent studies 1,ul volumes of serum were dispensed into wells, using 5-,l disposable capillary pipettes calibrated in 1pI increments. Plates were placed in a moist atmosphere at 4°C overnight and then transferred to 35°C for 4 h. Diameters of hemolysis zones were determined with the aid of a 7 x magnifying measurer and converted to areas, correcting for the area of the well. A ratio of 1.5-fold between areas of any two hemolysis zones was considered as indicating a significant difference in antibody concentrations of the sera tested. This ratio of areas corresponded to a difference in diameter of about 0.5 mm for small (3.5 mm) zones, or 1 mm for large (6 mm) zones, which in practice represented the smallest increase in size that could be objectively measured when comparing an acute-phase (or prevaccination) serum with its corresponding convalescent-phase (or postvaccination) serum. Conventional serological tests. HI tests, with sera treated with receptor destroying enzyme (Center for Disease Control reference reagent), and CF tests were performed by standard procedures (3, 17). NI tests were conducted with fetuin substrate in a manner similar to that described by Kendal et al. (8) but with twice the previous fetuin concentration and 3.2-fold or 4-fold serum dilutions. Animal sera. Ferrets were infected intranasally with 1 ml of undiluted allantoic fluid and bled out 12 to 14 days later. Human sera. Human sera were from two sources. Prevaccination and postvaccination sera were from adult volunteers immunized in September 1975, with a 1974 to 1975 formulation killed whole virus vaccine (Merck, Sharp and Dohme "Fluvac"), containing 700 chick cell agglutinin (CCA) units of A/Port Chalmers/1/73(H3N2) and 500 CCA units of B/Hong Kong/5/72. Vaccine was administered subcutaneously, and postvaccination sera were collected 3 weeks later. Serum pairs from persons naturally infected with influenza virus or other viruses had been submitted for diagnosis to the Respiratory Virology Branch from 1973 to 1975. Such sera were from patients with a clinical upper respiratory infection. Bleeding intervals were not uniform, but each convalescent-phase serum was collected at least 3 weeks after the acute-phase serum. All sera had been stored frozen at -20°C. No preservatives were added.

RESULTS (i) SRH for influenza A hemagglutinin antibodies, measured with recombinant virus antigens. To determine whether SRH effectively measured hemagglutinin antibodies under conditions where activity by neuraminidase antibodies was excluded, we prepared SRH plates with recombinant antigen A/Port Chalmers/1/73(H3)-equine/Prague/1/56(Neql) and tested with animal sera and human sera lacking antibodies to the Neql neuraminidase. Figure 1A illustrates the dose-response relationship when serum from a ferret infected with A/

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FIG. 1. Detection of influenza hemagglutinin antibodies by SRH: dose-response relationship. SRH plates contained sheep erythrocytes coated with recombinant virus AlPort Chalmers/i /73(H3)-equinel Prague/l 156(Neql). Hemolysis areas produced by dilutions of (A) serum from a ferret infected with A/ Port Chalmerslll73(H3N2) virus; and (B) convalescent sera from two people naturally infected with A/ Port Chalmers/i /73-like virus.

Port Chalmers/1/73 was tested at various dilutions. This test demonstrated that at high antibody concentrations a linear relationship existed between the area of hemolysis produced and the serum dilution. As the antibody concentration decreased, however, this relationship changed. At serum dilutions producing hemolysis areas smaller than about 15 mm2, the sensitivity of the SRH test to serum dilution was much less than at serum dilutions producing hemolysis areas of greater than about 15 mm2. A similar effect was observed with other animal sera (not shown) and also with convalescent-phase human sera from patients infected with A/Port Chalmers/1/73-like virus (Fig. 1B). When animal or human sera having "high" activities (hemolysis area >20 to 90 mm2 in the SRH test) were tested on three different days with gels prepared from different batches of erythrocytes, the standard error of results in the SRH test was 1 to 4%; this degree of reproducibility is similar to that obtained by Russell et al. (13). (ii) SRH for influenza B hemagglutinin antibodies. Since influenza B neuraminidases fall

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within one antigenic subgroup (6, 12), it was not possible to use an influenza B recombinant virus with an irrelevant neuraminidase. SRH plates were therefore prepared with the representative B/Hong Kong/5/72 whole virus antigen. Plates were tested initially with ferret serum to the virus B/Hong Kong/7/75, which is closely related to B/Hong Kong/5/72. The clearest patterns of hemolysis were obtained by preparing SRH plates with only about one-half of the erythrocyte concentration of that in plates made with influenza A viruses. Under these conditions, the ferret serum produced readily measurable clear zones of hemolysis, with a similar dose-response relationship to that shown for influenza A virus reactions (see Fig. 1). Additional analysis of the measurement of influenza B antibodies in human sera by the SRH technique is presented below. (iii) Evaluation of the SRH test for measurement of influenza hemagglutinin antibody rises in human sera. Paired sera selected for testing were from influenza vaccine recipients and from persons naturally infected with influenza viruses. Vaccinees had been immunized in 1975 with a vaccine containing A/Port Chalmers/1/73 killed virus and thus might be expected to produce antibodies to the prior A/ Hong Kong/8/68 strain as well as to the A/Port Chalmers/1/73 strain (10). Therefore, HI and SRH tests were conducted with both A/Hong Kong/8/68 and A/Port Chalmers/1/73 antigens. In all cases the antigens used in the SRH test were recombinant viruses, thus excluding any effects of neuraminidase antibodies. Table 1 shows that in 67% of the cases examined there was a total agreement between the HI and SRH results for the frequencies of antibody rises to both A/Hong Kong/8/68 and A/Port Chalmers/ 1/73 antigens. Furthermore, in 57 cases where an HI rise to A/Port Chalmers/1/73 was found, 52 (91%) also showed an SRH rise to A/Port Chalmers and, including A/Hong Kong/8/68 antigen, added little to the ability of either test to measure overall rates of seroconversion in response to the A/Port Chalmers vaccine. In a few instances where the HI test did not demonstrate a fourfold antibody rise, we observed an experimentally significant rise in titer obtained by the SRH technique. These rises, however, were of low magnitude, implying that there is no fundamental difference between the sensitivities of the two tests when detecting rises in levels of hemagglutinin antibodies. Antibody response to the influenza B component of the vaccine was also compared by HI and SRH tests. The overall agreement between SRH and HI in measuring the seroconversion rate to influenza B in the vaccinees was 83%

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TABLE 1. H3 hemagglutinin antibody rises in 75 recipients of vaccine containing killed A/Port Chalmersll / 73 virus: comparison of hemagglutination inhibition (HI) and single radial hemolysis (SRH) tests Antibody rise by HIb Antibody rise by SRHa

A/Port Chalmers/l/73 and A/Hong Kong/8/ 68

A/Port Chalmers/1/73 only

A/Hong Kong! 1/68 only

Neither virus

2 (3) 3 (4) 10 (13) 35' (47) A/Port Chalmers/1/73 and A/Hong Kong/8/68 1 (1) 0 (0) 5 (7) 2 (3) A/Port Chalmers/1/73 only 0 (0) 2 (3) 2 (3) 0 (0) A/Hong Kong/1/68 only 10 (13) 0 (0) 2 (3) 1 (1) Neither virus a A 1.5-fold or greater increase in area of clear hemolysis zone (or from 0 to .4 mm2). Antigens used to coat erythrocytes contained neuraminidase Neql. b A fourfold or greater rise in HI titer (or from