Single radial haemolysis for the assay of antibodies to some haemagglutinating arboviruses*

Bulletin of the World Health Organization, 57 (6): 937-942 (1979) Single radial haemolysis for the assay of antibodies to some haemagglutinating arboviruses * MIHAI DUCA,1 EUGENIA DUCA,1 LIDIA IONESCU,2 & HAYDAR ABDALLA3 The single radial haemolysis reaction has been shown to be suitablefor the quantitative assay of specific antibody to West Nile and Sindbis viruses. Only 5 [sl of undiluted serum are needed for the test, which can be performed on crude preparations of antigen and without removal of nonspecific inhibitors. It is therefore a very simple procedure. Moreover, it appears to be more specific than the classical haemagglutination-inhibition tests. A simple, accurate, and rapid laboratory test for antibodies to togaviruses (2) would be of consider- able value in routine serological diagnosis and seroepidemiological surveillance. One of the main problems is serological cross-reactivity between members of the same antigenic group, as demon- strated by the haemagglutination-inhibition (HI) reference test (4) and the complement fixation (CF) test (3). Owing to technical difficulties, neither the classical neutralization test (3) nor the recently described tests using fluorescent antibody or solid- phase radioimmunoassay would be satisfactory for routine serological testing for these viruses. In this paper, we describe the results obtained in the assay of antibodies to West Nile (genus Flavivirus) and Sindbis (genus Alphavirus) viruses, using the single radial haemolysis (SRH) method as described by Schild et al. (13-14), with influenza virus haemagglutinins. The new SRH test proved to be particularly useful for routine serological tests for togaviruses since it is not necessary to remove nonspecific inhibitors as in conventional HI tests, it requires only 5 ,ul of the undiluted serum specimen, and it can be performed using crude preparations of antigen with chicken erythrocytes. * From the Faculty of Medicine, Institute of Medicine and' Pharmacy, 6600 Iasi, Romania. This work was supported in part by a grant from the World Health Organization under Agreement V5/ 181/75. 1 Professor. 2 Chemist. Student. MATERIALS AND METHODS Virus strains The West Nile virus, prototype strain Egypt 101, and the Sindbis virus, prototype strain EgAr 339, were obtained from the State Bacteriological Laboratory, Stockholm; the HyPr virus original strain, the Calovo virus strain 184, and the Tahynia virus strain 92, were obtained from the Institute of Virology, Slovak Academy of Sciences, Bratislava. The viruses were maintained in suckling Swiss albino white mice, using the intracerebral route. Immune sera These were prepared in guineapigs, white rats, white mice, or rabbits, by repeated intraperitoneal, intramuscular, and hind footpad injections of virus- infected suckling mice brains, the antiviral serum being collected after 4-6 weeks. Human sera Serum was collected from an adult aged 42 years who had had an inapparent infection with West Nile virus, strain Egypt 101, 16 years previously after a laboratory accident (8). Pooled serum obtained from 5 adults with naturally acquired dengue infection (7) was also used. Haemagglutinating antigens These were prepared by sucrose-acetone extrac- tion from the brains of suckling mice infected with West Nile virus and from the brains of suckling white rats infected with Sindbis virus (4, 5). 3911 - 937 - 938 M. DUCA ET AL. "Crude" haemagglutinating antigens Mashed infected brains of suckling mice or rats were suspended in borate-saline buffer (200 g wet weight per litre), adjusted to the optimum pH value of 6.6 for West Nile or 6.2 for Sindbis virus. Control antigens For use as controls, extracts and "crude" suspen- sions were prepared from brains of normal unin- fected animals, as described above. Haemagglutination-inhibition (HI) tests These tests were performed as described by Clarke & Casals (4); sera were extracted with chilled acetone and adsorbed with goose erythro- cytes prior to testing (6). Preparation of SRH immunoplates Suspensions of freshly washed chicken erythro- cytes (100 ml/litre) were made in phosphate- buffered saline (PBS) at pH 6.6 for West Nile virus or pH 6.2 for Sindbis virus. The antigen was added to the erythrocytes in the form of sucrose-acetone extract or as " crude" suspension, at a concentration of 10-20 haemag- glutinating units per ml of 10% erythrocyte suspen- sion.The suspensions were held at 4°C for 10 min to allow the virus to adsorb on erythrocytes. The unadsorbed virus was then removed by 2-3 repeated centrifugations at low speed, followed by resuspen- sion of the erythrocytes in fresh PBS. Usually, only the first supernatant fluid contained some free virus. As controls, erythrocytes treated with uninfected mouse brain suspension were used. Immunoplates were prepared by incorporating 0.3 ml of virus-treated erythrocytes and 0.1 ml of guineapig complement in 2.6-ml volumes of melted agarose held at 45°C in a water-bath. The final concentration of complement in the gel was equiv- alent to a minimum of 2.5 haemolytic doses, mea- sured in a standard assay system with sheep erythro- cytes (1); fresh or conserved complement may be used (12). The agarose used was A-37 Indubiosea at a concentration of 15 g/litre in PBS adjusted to a final pH of 7.2, preserved with sodium azide (1 g/litre). After being vigorously shaken, the erythrocyte suspension in melted agarose was poured onto microscope slides in volumes of 3 ml per plate. Wells 2.5 mm in diameter were cut in the gel. Prepared a Industrie biologique franqaise, Genevilliers, France. immunoplates may be stored at 4 °C in a humid chamber for up to 10 days before use. SRH test procedure The serum, previously heated at 60 °C for 20 min, was added to the wells in the immunoplates in volumes of 5 pl of undiluted or 1:10 serum. The plates were then transferred to a humid chamber and incubated at 37°C for 16 h before they were read. Sera treated to remove nonspecific inhibitors and nonspecific haemagglutinins, as well as untreated sera, were tested. The diameters of the zones of complete or partial haemolysis that developed around the wells were measured with a transparent graduated ruler, as in the usual single-radial immunodiffusion (Mancini) test. Control tests for nonspecific haemolytic activity were performed with plates containing complement and nonsensitized erythrocytes. RESULTS Accuracy and sensitivity of SRH reactions Potent reactions to West Nile and Sindbis virus were obtained with mouse, rat, and guineapig im- mune sera in SRH tests with chicken erythrocytes treated with the homologous virus. Fig. 1 illustrates Fig 1 SRH reactions with mouse (top), rat (middle) and guineapig (bottom) immune serum in an agarose gel immunoplate containing guineapig complement(final concentration 1 :30) and chicken erythrocytes(10% suspension) sensitized with West Nile virus(10-20 haemagglutinating units/mi). The clear areas represent zones of lysed erythro- cytes produced by antibody to surface viral antigen. Each serum was distributed in wells in serial two-fold dilutions, starting with 1 : 1. ASSAY OF ANTIBODIES TO TOGAVIRUSES the reactions of serial dilutions (starting with undi- luted serum) of these three immune sera to West Nile virus. The dose response in the SRH test showed that the size of the zones of haemolysis decreased regu- larly with increasing serum dilution. As shown in Fig. 2, a plot of log10 zone diameter against log2 serum dilution gave as straight-line relationship. 1,2 1,1 10. 0,9. 0,8 - 0,7. 0,6- 0,5 0,4 0 0~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~6 '.0 u' cN 00 - 0e- - 0 '0 0 0eococ of dIno Ir rCRecC,orc/of'he seram dll'1110fl (1092 Sc"/e) Fig. 2. Dose-response curve of serial dilutions of guineapig immune serum to West Nile virus. A plot of the logl0 zone diameter against serum dilution (1092 scale) approaches linearity. of 1:5120 (250 [&l) displayed in SRH a titre of 1:10 240, i.e., 5 1tl of the serum dilution gave a 3-mm diameter zone of clear haemolysis. Specificity of SRH test As shown in Table 2 and Fig. 3, in the SRH test immune sera to West Nile or Sindbis virus reacted exclusively with the chicken erythrocytes sensitized with the homologous virus. The diameter of the zones of haemolysis surrounding the wells contain- ing the homologous antiserum was directly propor- tional to the content of HI antibody. While mouse, rat, and guineapig immune serum gave clear zones of haemolysis, the rabbit immune serum gave a zone of "incomplete" haemolysis, still easily measurable. The SRH reactions were negative in the absence of complement, as well as with nonsensitized eryth- rocytes or erythrocytes treated with control antigen, i.e., uninfected brains. Table 2. Specificity of the SRH reaction with rat immune sera Diameter of haemolysis HI titre with: zone (mm) Antiserum in the SRH reaction with: against: West Nile Sindbis West Nile Sindbis virus virus virus virus West Nile virus 1:1280 - 13.0 - Sindbis virus - 1:2560 - 12.0 In estimating levels of antihaemagglutinin, the SRH test was found to be slightly more sensitive than the HI test with West Nile virus: as shown in Table 1, a guineapig immune serum with an HI titre Table 1: Correlation between the HI and SRH titres of West Nile immune sera raised in three different species Species HI titre' SRH titreb mouse 1:480 1:160 rabbit 1:1920 1:320 guineapig 1:5120 1:10240 'Dilution of serum (250 IlI) producing 50% inhibition of the haemag- glutination with 6-8 haemagglutinating units. bDilution of serum (5 IAI) producing a 3-mm diameter zone of clear haemolysis. Fig. 3. Specificity of the SRH reaction. The agarose gel contained erythrocytes sensitized with West Nile (WN) virus. The wells in the top row contained, in order, the following immune sera: WN (mouse), WN (rat), Den- gue (man), Tahynia (rat), and HyPr (rat). The wells in the bottom row contained, in order, the following immune sera: WN (rat), talovo (rat), WN (guineapig), WN(guineapig), and WN (rabbit). Only the wells containing WN immune sera are surrounded by zones of haemolysis. The human Dengue postinfection serum gave a negative SRH reaction; the rabbit WN serum gave a zone of "partial" haemolysis, still easily measurable. 939 M. DUCA ET AL. As shown in Table 3, in the SRH test with West Nile virus, animal immune sera to HyPr, Calovo, and Tahynia viruses were negative, as was human dengue serum. On the other hand, serum obtained from a subject who had had a laboratory infection with West Nile virus was positive. It should be mentioned that the pooled dengue human serum was positive in the HI test with West Nile virus at a titre of 1:160. SRH reactions and nonspecific inhibitors Normal sera and immune sera to West Nile virus, containing different amounts of nonspecific in- hibitors (up to 1: 81 920), were obtained from different animal species, including man, and assayed, in parallel, in HI and SRH tests, both with and without prior treatment for removal of the non- specific inhibitors. As shown in Table 4, and in Fig. 4, nonspecific inhibitors or nonspecific haemagglutinins present in the sera tested did not affect the SRH reaction. SRH reactions with "crude" preparations of antigen The specificity, accuracy, and sensitivity of the SRH reaction remained unchanged when, instead of sucrose-acetone antigen extracted by the method of Clarke & Casals (4), "crude" preparations of anti- gen were used, provided that the pH was strictly adjusted to the optimum value for adsorption of the virus on to the erythrocytes, i.e., 6.6 for West Nile virus and 6.2 for Sindbis virus. The SRH tests were negative with erythrocytes treated with "crude" preparations or extracts of uninfected brain. Table 3. Comparison of HI and SRH reactions (with West Nile virus) of immune sera against the homolog- ous virus and other arboviruses Antiserum Homologous Diameter ofAntiserum Homologous haemolysis zone against: raised in: inHItest inSRHtest(mm) West Nile mouse 480a 10.5 rat 320 9.5 rabbit 1 920 13.0 guineapig 5 120 11.6 guineapig 10 240 13.2 human b 320 5.5 Dengue humanc 160 - Tahyina rat 140 - HyPr rat 120 - talovo rat 240 - aReciprocal of the dilution representing the serum titre. bAfter inapparent infection contracted in the laboratory (8). CAfter natural infection, pooled serum (7). Table 4. Comparison of HI and SRH reactions (with West Nile virus) of different sera (West Nile immune and normal sera) containing nonspecific inhibitors (NIH) and nonspecific haemagglutinins. Titre' in HI test Diameter of haemolysis zone(mm) in SRH test Titre of Serum tested nonspecific in serum in serum in serum in serum haemagglutinins with NIH b without NIHc with NIH b without NIHc guineapig immune 10 240d 10 240 12.5 12.8 normal 10 240 <10 - - - rabbit immune 20 480 1 920 5.1 5.1 160 normal 5120 <10 - - 160 human immune' 40 960 240 4.0 4.0 - normal 10 240 <10 - - 80 mouse immune 2 560 480 7.2 7.0 - normal 1 280 <10 - - rat immune 10 240 320 6.1 6.0 normal 7 680 <10 - - a Dilution of serum (250 R&I) producing 50% inhibition. b Untreated serum. c Treated with acetone as described by Clarke & Casals (4). d Reciprocal of the dilution representing the serum titre. @ After inapparent infection contracted in the laboratory (8). 940 ASSAY OF ANTIBODIES TO TOGAVIRUSES Fig. 4. SRH reaction and nonspecific haemagglutina- tion inhibitors. The agarose gel contained erythrocytes sensitized with West Nile (WN) virus. The clear areas represent zones of lysed erythrocytes. The wells in each pair of neighbouring rows con- tained, in the upper row, 5 >tl of a serum extracted with acetone for removal of nonspecific inhibitors and, in the lower row, the corresponding untreated sample of the same serum. Independently of animal species or content of non- specific inhibitors, only antisera against WM virus produced a zone of haemolysis (see Table 5) with a diameter related to the titre of specific antibody. DISCUSSION The single radial haemolysis (SRH) reaction was developed by Schild et al. (13-14) using influenza virus haemagglutinin. The test has recently been employed by Duca et al. (9), among others, for antigenic characterization of recent influenza iso- lates, and has been extended to some other influenza virus antigens by Haaheim (10). It has also been applied to some other haemagglutinating viruses, namely to rubella virus by Schild & Bradstreet (unpublished data, cited in reference 14) and to mumps virus by Strulovici & Copelovici (15) and Munziger & Novak (11). The present paper shows that the SRH reaction can also be used for the quantitative assay of specific antibody to West Nile and Sindbis viruses. The accuracy of the SRH test with these arbovir- uses was evaluated by comparing the SRH test and the HI test results and was found satisfactory. It has been demonstrated that, when the SRH reaction is calibrated for a certain species, 5 pd of undiluted serum are sufficient to enable the titre of specific antibody in the tested sample to be esti- mated from the diameter of the haemolysis zone. Although the specificity of the new SRH test has not yet been completely investigated, it is certainly higher than that of the classical HI test since, as shown in Table 4, the dengue pooled human serum and the HyPr rat immune serum, both of which belong to the Flavivirus genus and are consequently positive in the HI test with West Nile virus, were consistently negative in the SRH test with West Nile virus. In contrast, only the homologous immune sera, i.e., the human serum against West Nile virus (laboratory infection) and all animal immune sera raised with that virus, were positive in both the HI test and the SRH test. The simplicity of the SRH test considerably en- hances its practical value for the serological diag- nosis and surveillance of arboviruses. It can be performed with undiluted and untreated sera and it works well with erythrocytes sensitized with crude preparations of haemagglutinating antigen. The fact that the SRH test is unaffected by nonspecific inhibitors present in the serum (8) constitutes a considerable advantage for large-scale seroepidemiological studies. Since the immunoplates can be used for up to 10 days if kept at 4 °C in a humid environment, the SRH method is potentially suitable for sero- epidemiological field studies of arboviruses. No haemolysis with chicken sera has so far been detected in SRH tests using guineapig complement. This is an apparent drawback in view of the part played by birds in the ecology of these viruses. Possibly, the use of complement of other species would extend the applicability of the SRH test to these hosts. UMil LE TITRAGE DES ANTICORPS ANTI-ARBOVIRUS HEMAGGLUTINANTS PAR HEMOLYSE RADIALE SIMPLE Le present article decrit une reaction d'h6molyse radiale virus grippal par Schild et coil. Bien que tous les para- simple applicable aux virus West-Nile et Sindbis et qui metres n'en aient pas encore ete etudies, il est d'ores et s'execute facilement d'apres la technique utilisee pour le deja evident que de par sa precision, sa reproductibilite, sa 941 942 M. DUCA ET AL. sensibilite et sa specificite, cette reaction permet de d6tec- ter et de titrer de faqon tout a fait satisfaisante les anticorps diriges contre les antigenes de surface des deux virus. En ce qui conceme les taux seriques d'anti-hemagglutinines, la nouvelle reaction s'est montree legerement plus sensible et beaucoup plus sp6cifique que la reaction classique d'inhibi- tion de l'hemagglutination. II n'est pas non plus necessaire d'eliminer les inhibiteurs non specifiques comme c'est le cas pour la reaction d'inhibition de 1'hemagglutination. Avec le complement de cobaye, on peut utiliser aussi I'antigene viral brut et des hematies de poulet. REFERENCES 1. BRADSTREET, C. M. P. & TAYLOR, C. E. D. 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