key: cord-0004882-1xey8x79 authors: D'Ambrosio, E.; Battaglia, M. F.; Midulla, M. title: Single radial haemolysis for the determination of antibody to reoviruses date: 1981 journal: Arch Virol DOI: 10.1007/bf01314587 sha: fd5b793c46374cb6c07cdac40021ec53e36a94d8 doc_id: 4882 cord_uid: 1xey8x79 A single radial haemolysis (SRH) for reovirus antibody determination was developed and compared to standard haemagglutination-inhibition (HI) and complement fixation (CF) techniques. SRH appeared more simple and sensitive than CF, but less sensitive and less specific than HI. Single radial haemolysis (SRH) was originally developed for measuring antibody to influenza (9, 10), but has been successfully applied to other haemagglutinating and non-haemagglutinating human viruses (2, 3, 5--7, 14, 15) . Quantitation of antibody to reoviruses in human and animal sera has been usually performed by H I or neutralization (11, 12) , CF being group-specific and of low sensitivity. In this paper we describe the detection of reovirus antibody by a newly developed SRIt technique, in comparison with HI and CF (13) . Human O red blood cells (HRBC), without any coupling agent, were used. Freshly prepared "aged" chromium chloride-treated (1), gtutaraldehyde-stabilized (4) or "aged" chromium chloride-treated glutaraldehyde-stabilized HRBC (5) was no better than untreated ery~hrocytes. Sheep red blood cells, if treated with chromium chloride, could be substituted for ItRBC, but a few sera produced non-specific haemolysis, which never happened with HRBC. Reovirus type 1 (Lung), type 2 (D5 Jones) and type 3 (Abney) were grown on monotayers of monkey kidney cell lines (CV-t, LLC-MK, Vero) in serum-free Eagle's Minimal Essential Medium; the cells were disrupted by sonication. Antigen (HA titre 160) was mixed with an equal volume of 5 per cent ItRBC suspension in PBS and incubated at room temperature for 30 minutes; the erythrocytes were then washed 3 times and resuspended at 5 per cent in PBS. Each empty ttyland immunoplate received a mixture of 0.3 ml antigen-coated HI~BC suspension, 0304-S608/81/0068/0309/$ 01.00 E. D'A~BI~OSlO, M. F. BATTAGLIA, and M. MIDULLA: 0.I ml normal guinea pig serum (as a source of complement), and 2.6 ml molten 1.5 per cent agates• (Indubiose A 37) in PBS (10) . Five F1 undiluted heat-inactivated serum was added to 2 m m wells a n d the plates were i n c u b a t e d overnight at 37 ° C: a clear haemolysis zone of ~>3 m m diameter was considered a positive reaction. Wells filled with PBS in re•virus S R H plates, and plates prepared with H R B C incubated with s u p e r n a t a n t of sonieated uninoeulated cells served as negative controls. Of the 32 post-immunization samples from animals receiving a n y re•virus type, a n t i b o d y response was detected b y CF in 24 (75.0 per cent), b y S R H in 26 (81.2 per cent) and b y H I in t00 per cent,. Table 1 shows that, the results with St{H were less specific t h a n those with H i l l e r example the sera of animals i m m u n i z e d with type 3 did not react by H I with type 1 or 2, but 3 of 5 reacted by S R H and CF. W h e n 68 r a n d o m l y selected h u m a n sera were tested (Table 1) , S R H detected, as for guinea pig sera, m a i n l y group-specific antibody, b u t was more sensitive t h a n CF. H I detected a n t i b o d y in 57 (83.8 per cent), SIgH in 36 (52.9 per cent), a n d CF in 17 (25.0 per cent.) sera. W h e n the haemolysis ring Type 1 b 5 4 3 2 5 5 0 3 3 1 0 0 Type 2 b 2 3 3 5 5 5 0 3 3 0 0 0 Type 3 b 3 3 3 4 5 5 5 3 3 0 0 0 Each group of 5 animals was inoculated intranasally (8) with live re•virus type 1, type 2, or type 3. Sera were drawn after 5 and 6 weeks; results were identical except that SIgH zones were slightly larger at 6 weeks b These animals were sere-positive by HI against type 2 before inoculation diameters were plotted against CF titres (Fig. 1 ) , a good correlation was observed between the two techniques; similar results (not shown) were obtained for guinea pig sera. It, is n o t clear why the a n t i b o d y response b y SRH, using haemagglutin a t i n g antigen attached to RBC, appears different from t h a t measured b y H I --u s i n g influenza virus the results are very similar though anti-neuraminidase a n t i b o d y can be detected as well as III. Since all sera were pre-treated with kaolin, it is unlikely t h a t H I was detecting non-specific inhibitors. S R H is nevertheless a useful technique for work with reoviruses. I t is simple, uses small quantities of heat-inactivated serum, without need for dilutions, a n d is easy to read; in addition, the plates can be fixed with formaldehyde (9) or glutaraldehyde as a p e r m a n e n t record. I t might be applied to n o n haemagglutihating h u m a n viruses, such as herpesvirus instead of CF. The chromic chloride method of coupling antigens to erythroeytes: definition of some important parameters Haemolysis-in-gel and neutralization tests for determination of antibodies to mumps virus Evaluation of the hemolysis-in-gel test for the screening of rubella immunity and the demonstration of recent infection Preparation of conjugated erythrocytes for long term use in hemolytic plaque assays, complement fixation studies, or passive hemagglutinations: a comparative study of several methods Quantitation of antibody to nonhemagglutinating viruses by single radiM hemolysis: serologieM test for human coronavirus Application of single.radial-haemolysis test for the detection of antibodies to togaviruses Antibodies to human coronavirus 0C43 measured by radial haernolysis in gel Manual of clinical Microbiology A single radial haemolysis technique for the measurement of influenza antibody Si@e-radial-haemolysis: a new method for the assay of antibody to influenza haemagglutinin Microneutralization test for the reoviruses. Application to detection and assay of antibodies in sera of laboratory animals Antibody assays for enteroviruses and reoviruses Application of a microtechnique to viral serological investigations Determination of mumps and influenza antibodies by haemolysis-in-gel Demonstration of antibodies to measles virus by hemolysis-in-gel (HIG) test. Scand