key: cord-271884-86yl9ren authors: Traavik, T. title: Development of a modified immunoelectroosmophoresis method for Uukuniemi and Runde virus serology date: 1977 journal: Arch Virol DOI: 10.1007/bf01314789 sha: doc_id: 271884 cord_uid: 86yl9ren In search for a suitable method for sero-ecological screenings for arboviruses in Norway, efforts were undertaken to make the immunoelectroosmophoresis technique more sensitive than here to fare in detection of antibodies. The aim was to make it comparable to haemagglutination inhibition test in sensitivity, retaining the advantages in specificity, simplicity and capacity. This has been achieved by: 1. Using concentrated virus as antigen. 2. Performing electrophoresis in a gel consisting of an agar-agarose mixture in optimal concentrations. 3. “Sandwiching” the first specific electrophoretic run with an anti-species antiserum to the tested sample. The isolation of Uukuniemi (UUK) group viruses and a new coronavirus-like agent, Runde virus, from Norwegian Ixodes ticks (19, 21, 22, 23) , were intended to be followed up promptly by sero-ecological surveys by a standard haemagglutination inhibition test (HAI). It was soon decided, however, that certain disadvantages of HAI, with regard to our specific needs, would make it worthwhile to search for another screening method. After having solved the problem of making potent HA antigens for our weakly haemagglutinating viruses (24) , the remaining disadvantages of HAI were linked to the question of unspecific serum inhibitors, and to its broad group reactivity. We intended to perform sero-eeological screenings comprising human beings, small mammals, passerine birds, sea birds, wild ruminants, domestic animals and T. Tt~AAvI~: : reptiles. However, little is known about the qualitative and q u a n t i t a t i v e contents of serum inhibitors in m a n y of the groups above. Therefore it was desirable to use a serological screening method which eliminated the inhibitor problem b u t was comparable to H A I in sensitivity. The use of the immunoelectroosmophoresis (IEOP) technique has attracted increasing interest in virus serology due to its speed, simplicity and modest requirements for reagents. B u t its use has been hampered b y the lack of sensitivity, which is partly due to the anodal m o v e m e n t of most immunoglobulins under the conditions previously employed (12) . This paper reports how the adoption of relatively simple measures makes I E O P equal to H A I in sensitivity for detection of antibodies to Norwegian arbovirus strains. Since it eliminates the serum inhibitor problem and also has various praetieM advantages over HAI, it has become our standard screening method. The original isolations of the five virus strains used in these experiments are described elsewhere (22, 23) . They were isolated from suspensions made from lxodes ticks by intraeerebral inoculations of 1--~3 days old baby mice [Born: NMRI (SPF)]. SF E 1 and By E50 are Uukuniemi (UUK) group viruses from I. ricinus ticks. Ru E 82 is an U U K group virus from I. uriae, while Ru E81 and t~u E85 are coronavirus-like agents isolated from I. uriae. The two latter strains seem identical and we have tentatively termed them Runde virus. Ru E 82 differs antigenieally from the two other U U K viruses. Tahyna virus (strain 181) was kindly provided by Dr. V. Danielova, Institute of Parasitology, Czechoslovak Academy of Sciences, Prague as a 10 per cent lyophilized suckling mouse brain suspension from the 3rd passage. Tribee and Tickborne encephalitis (TBE) virus were kindly placed at our disposition by Dr. M. Gresikova, Institute of Virology, Slovak Academy of Sciences, Bratislava. Ten per cent lyophilized suckling mouse brain suspensions from the 13th and the 63rd passage, respectively, were used. The following kinds of antigenic preparations have been utilized in these experiments : Crude suckling mouse brains (SMB antigen) are 20 per cent suspensions of infected suckling mouse brains in PBS pH 7.4 with 0.75 per cent bovine Mbumine (APBS). Suerose-aceton extracted suckling mouse brains (SA antigen) were prepared according to CLARKE and CASALS (5), but the finM lyophilization step was omitted. Cell culture antigens (BHK antigens) were prepared by growing the viruses in BHK21/c 13 cells in Roux bottles. The infectious culture fluids are concentrated 100 to 300 times by precipitation with 6 per cent polyethylene glycol 6000/2.2 per cent NaC1 at pH 7.6 and 4 ° C (14) overnight, followed by lowspeed centrifugation and resolution of precipitates in APBS. Finally sonication is performed. For TBE, HeLa Bristol cells were used instead of BHK. It was found beneficial to include lipoprotein-absorption by the colloidal silieagel Aerosil (Degussa, Frankfurt a. Main) for M1 antigens (20, 24) . This was done in order to avoid lipoprotein deposition around the electrophoresis wells. This may in some instances interfere with the interpretation of specific precipitation lines. Uninfected mouse brains and BHK-cultures were treated in parMlel and used as control antigens. Each mouse initially received between 104 and 105 BMLDs0 (baby mouse lethal doses) in 0.25 ml brain suspension mixed with an equal volume Freund's complete a d j u v a n t (Difco) intraperitoneally. Thereafter 3 weekly injections without a d j u v a n t were given. After another week, an injection identieM to the first one was performed. Seven to 8 days later paracentesis and bleeding from the retroorbitM sinus were done. Reference mouse immune aseitic fluids to U U K (strain S 23), T B E (RSSE) and Tribec had been obtained from the Yale Arbovirus Research Unit. Rabbits received 3 injections of virus grown on BI-IK cells, concentrated 100 times by PEG/NaC1 and sonieated. They first received 5 ml intraperitoneally, and then 2 ml intravenously 2 and 4: weeks later. The rabbits were bled by cardiac puncture before the immunization started, and 2 weeks after the last injection. The antisera were adsorbed with packed B H K cells. All sera were inactivated at 56 o C and stored at --20 ° C. The antispeeies sera used in these experiments were R a b b i t antimouse Ig (RAM/Ig) and R a b b i t anti-human Ig (RAHu/Ig) from Nordic Immunol. Lab., Tilburg, the Netherlands. Human Sera The h u m a n sera were selected from the files of the Virus Laboratory, H a u k e l a n d hospital, Bergen. The sera had been remitted for all kinds of suspected virological diseases and controls. Our only criterion for inclusion was that the patient should live in or near by areas infested by 1. ricinus. The technique is a modification of the m e t h o d of AnTER. et al. (1) , employing tile commercial I-Iepascreen eleetrophoresis apparatus (Spectra Biologicals, Oxnard, California). For the electrode trays as well as for the gel a barbital buffer with an ionic strength of 0.050 and a p H of 8.2 (Spectra Biologieals) is employed. Gels are made on precoated lantern slides, 8 × 8 cm. Twelve ml moulten 1 per cent agarose are poured on to each slide, giving an average gel thickness of 1.9 ram. Three double-rows of 10 wells are cut in the gel. The well diameters and interdistances are 3 ram. Two slides are run in the apparatus at the same time. R u n n i n g time is 60~ 90 minutes. The reagents are applicated by the aid of a 10 ~1 Oxford automatic pipette. I n screenings, this technique permits the simultaneous testing of 58 unknown samples. The following paramters have been tested: a) The optimal antigen preparations and concentrations. b) The composition of gels. Mixtures of Baeto agar (Difco) and agarose (l'Industrie Biologique Frangaise ) from 0.1--0.9 to 0.9--0.1 per cent have been used. c) The effects of antispeeies antiserum. After the termination of the first electrophoresis run, the antiserum well was filled with antispeeies (anti-mouse or anti-human Ig) and the electrophoresis was continued for 3 0~6 0 minutes. This sandwiching should theoretically trace immuneeomplexes which are not big enough to be read directly as precipitates. d) The effects of varying well-diameters and -interdistances. When screening unknown samples, the specificity of I E O P precipitation lines was secured b y the sensitive gel precipitation method (CHI) (closed hexagon immunodiffusion) (18) . Antigens were allowed to diffuse for 8 12 hours before filling of antisermn wells. R e p a t e d fillings of wells were necessary in some instances. W h e n needed, sera were concentrated by polyaerylamide gel (Lyphogel, Gelman Instr. Co.) (2) . I n all I E O P runs, positive and negative controls for antigens as well as antisera were included. (5), using microtitration equipment (Cooke Engineering Co., Alexandria, VA). For U U K viruses optimum conditions were pit 5.6 at 37 ° C, for t~unde virus pill 6.4 at 4 ° C (22, 23) . CFT was performed by standard procedures (10) using 2 units of complement in the final test,. The optimum antigen dilutions used for antibody detection was found by checker-board titrations. I n 1 per cent agarosc gel, crosstitrations showed that all 3 types of antigens had to be diluted to obtMn maximal sensitivity in antibody-detection. The celt culture antigens had the highest titers, and also effected a 2--4-fold increase in the titers of the antisera. Sandwiching the reactions with rabbit anti-mouse serum gave only a two-fold rise in antibody titers. These results are illustrated in Table 1 . I n contrast, if an mlbalanced system is encountered, i.e. the antigen is too strong to attain optimal sensitivity, the effect of antispecies serum is much more striking, on occasions it has increased the titer of antiserum b y a factor of 8. A remarkable higher sensitivity was demonstrated by mixing increasing amounts of ~gar into the electrophoresis gel. The concentration of antigen needed for optimM conditions had to be elevated in parMlei to the relative agar-concentration. B y using a gel composed of 0.4 per cent agar and 0.6 per cent agarose, and concentrated B H K antigens, results comparable to the H A I titers were obtained for the reference sera with all the viruses tested. This is demonstrated in Table 2 . The reasons for the improvement was clearly seen b y immnnoelectrophoresis as demonstrated in Figure 1 . With increasing agar concentration in the gel, more of the antibodies move cathodically, but at the same time the anodal movement of the antigens are hampered, so that more concentrated antigens are needed. Under balanced conditions, i.e. near to equivalency, the effect of "sandwiching" is not very pronounced, raising end-point titers b y 1--2 dilution steps a Antibody titers are given as reciprocal titers b Ag--antigen, ab--antibody Rabbit antimouse Ig (Tables 1, 2) . However, when unbalanced systems are met with, "sandwiehing" is improving the results considerably (Table 2) . The ability to detect antibodies and antigen increased to some e x t e n t with dilution of antigen and a n t i b o d y as d e m o n s t r a t e d b y checker-board titrations. (Figs. l b and 1 d) . In Figures I a and 1 b mouse hyperimmune sara were applieated in the wells, and eleetrophoresis was performed for 90 minutes. The corresponding viruses, SA antigens, were applieated in the throughs, and diffusion took place overnight. In Figures 1 e and 1 d antigens were eteetrophoresed and the corresponding antisera applieated in the throughs. The viruses used ware from above: By E 50, I{u E 82 and I{u E81 A marked prozone was seen for all viruses. The prozone effect could be restricted to some degree b y "sandwiching", b u t the effectiveness of this varied from one experiment to another, and could no~ be relied upon. Consequently, the optimal dilution of antigen is considered the one giving precipitation lines for all antibodydilutions, discriminating the question of absolute end-point titer. These features are illustrated in Figure 2 . Increasing the well diameter to 4 m m a n d varying the interdistances between 3 a n d 8 m m gave no benefit in a n t i b o d y detection. Increased interdistanccs effected the optimal r u n n i n g time adversely. Some sera which had been found sero-positive to U U K virus in H A I were titrated in parallel in H A I , the original I E O P a~nd I E O P with 0.4 per cent agar to 0.6 per cent agarose. The results are shown in Table 3 . There was no correlation Antibody titers given as reciprocal values b Concentration of agar mentioned first e Rabbit antihuman Ig in a second electrophoresis run Table 4 Antibody preparation b) The precipitating antibodies of the antisera and mouse aseitic fluids could be adsorbed by using homologous virus-antigens, but not by using any hegerologous antigen. e) When anti-human antiserum was used to sandwich reactions with mouse antisera or immune ascitic fluids, and anti-mouse antiserum was used to sandwich human sera, no increase in tigers was seen. Neither anti-human nor anti-mouse angisera could improve the results obtained by rabbit sera. d) The UUK group viruses By E 50 and Ru E 82 had earlier displayed a oneway serological relationship in CHI (22) . Antiserum to By E50 reacted with both viruses, while antiserum to I{u E 82 reacted with the homologous virus phenomenon was seen also in IEOP. During the past few years, the IEOP method has found its main application in the detection of hepatitis B surface antigen (HBsAg) (t, 7, 16, 20) . It also has been used to some extent in the diagnosis of certain bacterial (6) and fungal infections (8) . In virology, the IEOP method has been employed in studies of myxoviruses (11), poxvirus (13), herpes simplex (25) and some plant viruses (17) . Recently, TSOTSOS (25) examined the possibilities of using IEOP for typing and subtyping of various viruses belonging to major groups. However, very few researchers have investigated the possibility of using IEOP as a method for detection of viral antibodies. The main advantages of IEOP over other serological methods would be its simplicity, speed, the possibility of testing simultaneously many specimens, and its modest requirements for reagents and equipment. Obviously, however, the assumption has been that this could not compensate for the insensitivity of the method in detection of. antibodies. Even so, IEOP modifications have been elaborated for screening of antibodies to influenza virus (4), California encephalitis virus (3), rubella virus (9) and measles virus (15) . Bug in all these cases, the sensitivity in antibody detection seemed inferior to the methods routinely used for these viruses. Even in the future, sero-eeological screenings wili be an integrated part of efforts to evaluate the extent and significance of arboviruses. The most commonly used technique for this purpose has been HAL In addition go the unreliability connected with unspeeifle inhibitors, HAI has certain other major disadvantages (apart from the fact that haemagglutinafion is not common to all arboviruses). The most serious objection to HAI as a sero-ecological tool is its very broad reactivity. Cross reactions occur between viruses which are not even transmitted by the same vector, but which might nevertheless circulate within the same areas. We have shown, for the Norwegian arbovirus strains, as has already been demonstrated for other virus groups (25) , that IEOP is virus type and even subtype specific, and thus offers advantages over the HAI test in this regard for sereecological screenings. The low consumption of serum is another very important factor. In many instances very limited amounts of blood can be drawn from each individual of animal species that are to be investigated, and one often wishes to perform tests against more than one antigen. The modifications which have been described in this paper to achieve a very sensitive IEOP test for antibody detection, are all simple and inexpensive. Some of the same effects have been obtained for hepatitis B diagnostics by employing discontinuous buffersystems (26) . As pointed out by KELKAI~ and NIPI~ADt~AR (t2) it is, however, a much simpler and cheaper solution to establish the optimal agaragarose mixture for a given antigen/antibody system. High-grade concentration of sera is a laborious and timeconsuming task, and often is prohibited by the sm~]l volumes of blood available. Provided the opportunity to concentrate the antigen, the modifications used in this work to obtain a sensitive I E O P for antibody detection might prove valuable also with other viruses. The relative failure experienced with TBE virus in these investigations m a y illustrate the problem of concentration. TBE has a considerably smaller molecular mass than the other viruses used, and according to TseTses (25) , this needs to be compensated for by the use of a more concentrated antigen. Counterelectrophoresis for detection of hepatitis associated antigen : Methodology and comparison with gel diffusion and complement fixation Hepatitis-associated antigen: Improved sensitivity in detection California arbovirus (La Crosse) infections. II. Preeipitin antibody tests for the dAagnosis of California Encephalitis A rapid method for demonstration of precipitating antibody against influenza virus by counterimmunoe]ectrophoresis Techniques for haemagglutination inhibition with arthropodborne viruses Immunologic investigations of meningococcal disease. I. Groupspecific l~eisseria meningitidis antigens present in the serum of patients with fulminant meningococcemia Rapid detection of Australia antigen by counterimmunoelectrophoresis Diagnostic royceserology by immunoeleetrophoresis Demonstration by irnmunoe]ectro~osmophoresis of precipits¢ing antibodies to a purified rubella virus antigen Arboviruses Immunochemical studies of influenza virus and associated host tissue components Agarose and counterimmunoeleetrophoresis Modified IEOP for Uukuniemi and Runde Virus Serology Detection of vaecinia antigen and antibody by counterelectrophoresis Concentration and purification of vesicular stomatitis virus by polyethylene glyeot "precipitation Determination of measles virus-specific nueleoeapsid antibodies by means of counterimmunoeleetrophoresis Immunoeleetrophoretischer Nachweis yon "Hepatitis-associated Antigen Immunoosmophoresis, a rapid and sensitive method for evaluating viruses A sensitive modification of the Ouchterlony technique. Detection of hepatitis associated antigen in a dosed hexagonal system The isolation of an agent related to Uukuniemi virns from Norwegian Ixodes ricin.us ticks. Acta path. microbioh Stand A method for production of antisera to hepatitis B antigen subtypes, and the distribution of subtypes D and Y among Norwegian hepatitis B patients. Acta path. microbioh Scand Tick-borne viruses in Norway Uukuniemi group viruses isolated in Norway Runde" vinls, a coronavirus-Iike agent associated with seabirds and ticks Improvement of arbovirus HA antigens by treatment with a colloidal silica gel and sonication Identification and typing of viruses and detection of antiviral antibodies by counterimInunoeleetrophoresis Enhanced detection of Australia antigen in serum hepatitis by discontinuous eounterimmunoelectrophoresis