Immune-mediated fever in the dog 167 sera from healthy controls, both groups com- prising different breeds. This corresponds well with what has been published by Hansson et al. 1996. Screening for antibodies against extractable nu- clear antigens (ENA) was done by 2 methods displaying partly overlapping results One tech- nique used immunelectrophoresis in agarose gel with calf thymus antigen (Calf thymus ace- tone powder 60 mg/ml, Pel-Freez, Rogers, AR). Litex agarose gel (FMC Bio Products, Rock- land, ME) and barbiturate buffer (0.05 M, pH 8.6) were used for electrophoresis and 10 µl of ENA reagent applied to 20 µl of undiluted ca- nine serum. The electrophoresis was run for 45 min using 120V and 44mA. Antibodies against ENA bind to antigen and create a visible band of precipitation in the gel. The other method used was an ELISA anti ENA screening kit (Quanta Lite™ Inova Diagnostics INC. St. Louis, MO) which is composed of 6 puriﬁed autoantigens, all well characterised in human diagnostics: SSA, SSB, RNP, Sm, Scl-70 and JO-1. The ELISA kit was modiﬁed for applica- tion with canine sera by using a rabbit antidog IgG peroxidase conjugate diluted 1:25000 (Sigma Chemical Co. St. Louis, MO), but oth- erwise following the procedure described for the kit which implies a serum dilution of 1:100. By the electrophoresis method 41 out of 141 patient sera (29%) were tested as positive whereas the result was 0/45 in the controls. Correspondingly the ELISA method gave 44 positive out of 129 sera (34.1%) and 1/45 in the controls: Positive reactions to all 6 speciﬁc ENA antigens could be detected among the positive ENA sera (unpublished). One technique was established for detecting an- tibodies to chromatin (DNP) using ELISA kit with puriﬁed antigen (Novamed Ltd. Jerusa- lem, Israel) and applying the same adaptation for canine sera as for the ENA ELISA kit. As substrate for detecting antibodies to native DNA by IIF was utilized a protozoon, Crithidia luciliae (Aarden et al. 1975). The crithidiae were cultivated in the laboratory and dispersed onto slides to be used in IIF. Like in the human variant a serum dilution of 1:10 was applied. The anti DNP test gave 53 positives out of 142 patient sera tested (37.3%) and 1/45 controls. The anti DNA method gave no positive reaction in any sera tested, which seems to correspond well with ﬁndings of other investigators (Hans- son et al. 1999, Monier et al. 1980, Thoburn et al. 1972). The activity of IL-6 was determined using IL-6 dependent mouse hybridoma cell line B13.29 clone B9 (Aarden et al. 1987, HogenEsch et al. 1995, Carter et al. 1999). Serial dilutions of the test sample were incubated for 72 h with IL-6 dependent cells. Viability was measured in a colorimetric assay with a tetrazolium salt (Sigma Chemical Co., St. Louis, MO) (Mos- mann 1983). rIL-6 (Brakenhoff et al. 1987) was included as a standard. The detection limit of assay was 15-20 pg IL-6/ ml serum. TNF-α was determined by cytotoxic effect on the ﬁbrosarcoma cell line WEHI 164 clone 13 (Espevik & Nissen-Meyer 1986, Hogen Esch et al. 1995, Carter et al. 1999). rTNF-α (Biogen, Cambridge, MA and BASF/ Knoll, Ludwigshafen, FRG) were included as standard. The detection limit of the assay was 2- 3 pg TNF-α/ml serum. An antiserum to rTNF (Neutralizing capacity, 600ng rTNF-α/ ml) (Es- pevik & Nissen-Meyer 1986) completely neu- tralized the TNF-α activity in the serum sam- ples. Results All patients responded to prednisolone therapy by abolition of clinical signs. A 4 months old female Irish setter in the muscle and joint group was euthanized because of 2 relapses, and that the owner preferred to start with another and healthy pup. This Irish setter became ill for the Acta vet. scand. vol. 43 no. 3, 2002