Dalsgaard, K.

Ohmann, H. B

University of Queensland eSpace

Acta vet. scand. 1980, 21, 705-707. 
Brief Communication 
INDIRECT IMMUNOFLUORESCENCE USING F(ab') 2-Il\IMUNO-
REAGENTS FOR THE DEMONSTRATION OF BOVINE VIH.AL 
DIARRHEA VIRUS (BVDV) ANTIGEN IN LYMPHOID TISSUE 
The immunofluorescence technique has proven a valuable tool 
in the investigation of viral replication in Yivo and in vitro. In 
our studies on the pathogenesis of BVD special attention was 
focused on antigen localization in lymphoid tissue and on the 
characteristics of virus replication in bovine macrophaf;es. In 
this connection it was considered momentous to be able to dif-
ferentiate between possible membrane-associated and C"{toplas-
malically located viral antigens. This led to considerat ions on 
possible non-specific binding of conventional immuP.ological 
reagents to Fe receptors on the cells. Receptors for the Fe portion 
of IgG haYe been described on bovine lymphocytes, polymorpho-
nuclear neutrophils and macrophages (Rossi & J{ie::el 1977, 
Grewal et al. 1978, Rothlein et al. 1980) . Moreover, receptors for 
IgG have been demonstrated in vascular endothelia of cattle 
(l(asukawa et al. 1980). 
IL has been claimed that surface membranes are def,troyed by 
normal fixation methods (Hijmans et al. 1969), but Biberfeld 
et al. (1974) showed that, although damage did oc,~ur during 
acetone fixation, it was not complete. Acetone made <:ells partly 
permeable to immunoglobulin, facilitating intracellular pene-
tration of FITC-conjugates, but left some cell surface antigens 
intact as revealed by incomplete elimination of specific staining 
of surface-bound immunoglobulins. We assumed that some Fe 
receptors might also pass this treatment undamag€' 3, especially 
when 20 % acetone solution was used for fixation to obtain a 
better preservation of morphology. 'Ve therefore concentrated 
on the use of antibodies where the Fe portion was removed by 
pepsin treatment. Initially, direct immunofluorescence using 
FITC-conjugated F(ab') 2-fraction of bovine anti BVDV hyper-
immune serum was tested, but the intensity wa·; too low for 
practical use. The & Fellkamp (1970 ) have shown that a direct 
correlation between the binding affinity in immu .10fluorescence 
and the precipitating activity of antibodies does ex ist. In contrast 
to porcine immunoglobulins, bovine immunoglc bulins possess 
rela tively poor precipitating activity (Dalsgamd, unpublshed 
observation) , and the activity may be further reduced after pep-
4, 
706 H. B. Ohmann et al. 
sin treatment in the production of F (ab' ) 2-fragments (.llpller 
197~1) . 
Amplification of Lhe system was finally achieved by the in-
direct method using FITC-conjugated F (ab' ) 2 anti species rea-
gent, and by the application of F (ab' ) 2-fragmenls from porcine 
hyp~rimmune serum against BVDV: 
H y p e r i m m u n e a n t i s e r u m a g a i n s t B V D V was 
raised in pigs by repeated intravenous injection of the Danish 
strain U g 59 ( Ollmann et al., in prep. ). 
R a b b i t a n t i s e r u m a g a i n s t s w i n e I g G was pre-
pared ~ssential ly by the method of Harboe & Ingild (1973). 
F (a b ' )'2 - fragments of swine and rabbit serum were pre-
pared a?~cording to Poulsen & Hjort (1980) using pepsin digestion 
of total serum protein. An enzyme protein ratio of 1: 50 was 
used overnight at pH 4/ 37°C. After centrifugation and dialysis 
the fragments were purified by gel chromatography on Ultrogel 
ACA 34: (LKB products). By SDS-polyacrylamide gel electro-
phoresis (Laemmli 1970) it was confirmed that the method 
yielded relatively pure F (ab' ) 2-fragments. The elimination of 
Fc-media~ed complement fixing activity was confirmed by the 
method described by Eskildsen (1975 ). 
FITC-conjugalion of rabbit F(ab' ) 2 was performed by 
Lhe method described for total JgG by The & Fellkamp. By the 
biuret method (I(abat & Mayer 1964) the extinction EiS~m of a 
1 % solution of F (ab' ) 2 was estimated at 8 and of intact IgG at 
12. F(ab' ) 2-conjugation products with an FITC/ Protein ratio 
about 50 % higher than described for intact IgG were used 
accordingly. Fluorescein isothiocyanale (FITC) isomer 1 (Balti-
more Biological Laboratories ) was used for the conjugation. The 
conjugates were stored in the dark at 4°C with 0.2 mg/ rnl NaN3 
added. 
Bovine macrophage cultures, unfixed or fixed in 20 % buf-
fered acetone, and appropriate cryostate sections, prepared as 
described elsewhere (Ohm ann et al. ), were stained by incubation 
for 60 min with F(ab') 2 swine anti BVDV (diluted 1: 80) fol-
lowed by 4 washings in PBS, 5 min each, and by 30 min incuba-
tion with the FITC-conj ugate (diluted 1 : 40 ). Reactions were 
run in moisl chambers at 37 °C. Three washings in PBS and 1 
in distilled water removed unreacted conjugate. Fluorescence, 
specific for BVDV antigen, was confined to the cell cytoplasm, 
often with highest intensity and a granular distribution in the 
juxta-nuclear region. In tissue sections, the specific reaction was 
easily distinguished from occasional yellowish auto-fluorescing 
-llelle Bielefeldt Olwl<Lll/1 & Kristian Dalsgaard: Bovine viral diarrhea IJirus. 
Fig u re 1. Bovine macrophage culture 8 days after inoculation of 
BVDV. Staining with swine F (ab') 2 anti BVDV followed by FJTC 
conjugated rabbit F (ab'h anti porcine IgG. 
Bovine viral diarrhea virus 707 
clumps, related to cell debris or to granulocyles. The non-specific 
staining of elastic and collagenic components seen in preparations 
stained by the application of whole bovine antisera was negli-
gible. 
ACKNOWLEDGEMENTS 
The excellent technical assistance of miss Helle Skplt is highly 
appreciated. 
Helle Bielefeldt Ohmann 
The Depm1ment of Pathology and Pathological Anatomy, 
Royal Veterinary and Agricultural University, Copenhagen, and 
](ristian Dalsgaard 
The State Veterinary Institute for Virus Research, 
Lindholm, Kalvehave, Denmark. 
REFERENCES 
Biberfeld, P., G. Biber[eld, Z. Molnar & A. Fagraeus: Fixation of cell-
bound antibody in the membrane immunofluorescence test. J. 
Inunun. Meth. 1974, 4, 135- 148. 
Eskildsen , M.: Complement fixation with porcine serum antibodies. 
Acta path. microbial. scand. Sect. C 1975, 83, 315-324. 
Grewal, A. S., B. T. Rouse & L. A. Babiuk: Characterization of surface 
1·cceptors on bovine leukocytes. lnt. Arch. Allergy 1978, 56, 
289-300. 
llarboe, N. & A. Jngild: Immunization, isolation of immunoglobulins, 
estimation of antibody titre. Scand. J. Immunol. 1973, 2, Suppl. 
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Hijmans, W., H. R. E. Schuit & F. J(lein: An immunofluorescence pro-
cedure for the detection of intracellular immunoglobulins. Clin. 
exp. Immunol. 1969, 4, 457- 472. 
](abat, E. A. & M. M. Mayer: Experimental Immunochemish·y. 2nd Eel., 
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Kasukawa, R., M. Okada & S. lgari: Rcceptors for IgG Fe and comple-
ment in the mammalian aorta. Int. Arch. Allergy 1980, 61, 175-
182. 
Laemmli, U. /(.: Cleavage of structural proteins during the assembly 
of the head of bacteriophage T4. Nature (Lond.) 1970, 227, 
680- 685. 
Mpller, .V. P. H.: Pc-medicated immune precipitation. l. A new role of 
the Pc-portion of IgG. Immunology 1979, 38, 631-640. 
Ohmann, H. B., M. Holm Jensen , ](. J. Sf}rensen & K. Dalsgaard: De-
monstration of bovine viral di-arrhea antigen in tissue sections 
by immunoperoxidase assay. In preparation. 
Poulsen, F. & T . Hjorlh: A simple method for the production of 
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Rossi, C. R. & G. K. Kiesel: Bo,rine immunoglobulin G subclass recep-
tor sites on bovine macrophages. Amer. J. vet. Res. 1977, 38, 
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Rothlein, R., D. W. Johnson & C. C. Muscoplal: Enumeration and 
characte1·ization of bovine blood, spleen and ly mph node cells 
containing immunoglobulins. Int. Arch. Allergy 1980, 61, 100-
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The, T. H. & T. E. W. Feltkamp: Conjugation of fluorescein isothio-
cyanate to antibodies. I. Experiments on the conditions of con-
jugation. Immunology 1970, 18, 865-873. 
( Received November 4, 1980). 
Reprints may be requested from: Dr. K. Dalsgaard, the State Veteri-
nary Institute for Virus Research, Lindholm, DK-4771 Kalvehave, Den-
mark. 


Indirect immunofluorescence using F(ab')2-immunoreagents for the demonstration of bovine viral diarrhea virus (BVDV) antigen in lymphoid tissue

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Indirect immunofluorescence using F(ab')2-immunoreagents for the demonstration of bovine viral diarrhea virus (BVDV) antigen in lymphoid tissue

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