Researchin VeterinaryScience1992,53, 393-396

Preparation and use of a monoclonal antibody to detect psittaci antigen in paraffin-embedded tissue sections

Chlamydia

E. BOLLO, B. BIOLATTI, A. DONN, Universitft degli Studi di Torino, Dipartimento di Patologia Animale, Via Nizza, 52-10126 Torino, Italy, C. TURILLI, Istituto Zooprofilattico Sperimentale delle Venezie Sezione di Padova, Via G. Orus, 2-35100 Padova, Italy, A. J. WILSMORE, Royal Veterinary College, Department of Animal Health, Boltons Park, Hawkshead Road, Potters Bar, Hertfordshire EN6 1NB

A murine monoclonal antibody prepared against an ovine abortion isolate of Chlamydiapsittaci (A22/Teramo) revealed specific binding to a 57 kDa chlamydiai antigen in immnnohlotting studies. The monoclonal antibody was able to detect intracytoplasmic chlamydial inclusions and scattered elementary bodies in infected McCoy cell culture, and on formalin-fixed paraffin-embedded tissue sections both from experimentally infected mice and from fetal membranes of cases of ovine enzootic abortion. C H L A M Y D I A E are obligate intracellular parasitic microorganisms responsible for a variety of diseases in both animals and man (Storz 1971, Ward 1983). The genus Chlamydia is divided into three species, C trachomatis, C psittaci and the newly recognised C pneumoniae (TWAR strain). Cpsittaci infects a wide variety of birds and mammals, causing several diseases such as enzootic abortion and polyarthritis in sheep, pneumonia and enteritis in birds, and conjunctivitis and encephalomyelitis in other domestic mammals (Shewen 1980). Monoclonal antibodies (mAbs) directed against different strains of C psittaci have been useful in characterising chlamydial epitopes (Toyofuku et al 1986, Seki et al 1988) and for classifying the immunotypes of isolates from birds and mammals (DeLong and Magee 1986, Fukushi et al 1987, Andersen and Van Deusen 1988, Takahashi et al 1988). The aim of this research was to produce a mAb against an ovine abortion isolate of C psittaci for detecting chlamydial antigen in formalin-fixed and paraffin-embedded infected tissues. The ovine abortion isolate A22/Teramo Cpsittaci was propagated in the yolk sac of seven-day-old chick embryos (Stamp et al 1950). Subsequent cultivation was performed on monolayer cultures of McCoy cells (Johnson et al 1983). Partly purified suspensions of tissue culture chlamydial elementary bodies were used for mouse immunisation and enzyme-linked immunosorbent assay (ELISA)screening of the hybridoma cell culture fluids. Cells were collected together with the culture fluid 48

to 72 hours after infection. After three cycles of freezing/thawing, the material was centrifuged at 15,000 g for one hour at 4°C, the supernatant discarded, and the pellet suspended in Dulbecco's phosphate buffered saline (PBS), pH 7.3. The antigen was passed through a 25 gauge needle, then centrifuged at 30 g for five minutes at 5°C. The pellet was discarded and the supernatant stored at -70°C. Female BALB/Cmice were immunised by intraperitoneal injection with 0-1 ml of the A22 antigen suspension, emulsified in 0.25 ml of Freund's complete adjuvant and 0-15 ml of PBS. Booster immunisations were administered 10, 20 and 21 days after the first injection. Murine mAbs directed against Cpsittaci were produced as described by Kohler and Milstein (1975). Briefly, 22 days after the first injection, the mice were killed and the spleens removed aseptically. Immune spleen cells were mixed with SP 2/0 - Ag 14 myeloma cells at a ratio of 10:1, centrifuged, and fused by treatment with polyethylene glycol 1500 (M. A. Bioproducts) at a final concentration of 50 per cent in Dulbecco's Modified Eagle's Medium (DMEM) over 45 seconds, followed by subsequent dilution with DMEM for one minute, and centrifuged at 300 g for five minutes. The cell culture was seeded in 0.2 ml volumes into 96-well microculture plates, incubated and allowed to grow for one week. Supernatants of wells with cell growth were screened for antibodies against chlamydial antigen in the ELISAdescribed below. The hybridoma ceils producing antibody against Cpsittaci were cloned twice by the limiting dilution technique to obtain a stable cell line. For the present study, one mAb coded 1-2 was selected, which was obtained in ascitic fluid after inoculation of 2 × 106 hybridoma cells intraperitoneally into BALB/Cmice treated with 0.5 ml pristane (2,6,10,14-tetramethylpentadecane; Sigma), according to the method described by Hoogenrad et al (1983). An ELISAtechnique was used to screen hybridoma cell culture supernatants, ascitic fluid and mouse antiCpsittaci polyclonal hyperimmune sera for chlamydial antibody. Microtitre plates (Dynatech) were incubated 393

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E. Bollo, B. BiolattL A. Donn, C Turilli, A. J. Wilsmore

for 16 to 18 hours at 37°C with 10 lag per well of chlamydial antigen in carbonate buffer (pH 9-6). After 200 blocking with 0.5 per cent bovine serum albumin in BSAfor one hour at 37°C, either undiluted tissue culture supematants, ascitic fluid or mouse sera at serial dilu116 tions in 0.05 per cent Tween 20 in PBS were added and the plates incubated for two hours at 37°C. The binding 97 m m "~ 97 of specific chlamydial antibodies was detected with horseradish peroxidase-conjugated goat anti-mouse IgG (Dakopatts) diluted 1:40 in PBs, and the plates were incubated for two hours at 37°C. The substrate solution was added and the plates were further incubated for 10 minutes. The reaction was stopped with 1 N sulphuric acid and the absorbance measured with a microplate spectrophotometer (Titertek Multiskan) at 450 rim. 45 ee =m 45 The antibody class of mAb 1-2 was found to be subclass IgG1 using the Mouse Typer Subisotyping ELISA Kit (BioRad). / Polypeptide profiles of the reduced chlamydial antigens were obtained by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) according g 31 to the method described by Laemmli (1970). SDS-PA~E was performed on 12.5 per cent polyacrylamide slab gels in tris-glycine buffer (pH 8-3) containing 0.1 per cent SDS at a constant current of 12 mA for 16 hours after samples had been heated for three minutes at FIG 1 : Immunobiotting patterns of 57 kDa chlamydial antigen detected 100°C in solubilising solution (0.01 M tris-hydrochloric with mAb 1-2. Lane 1 was applied with mouse anti-C psittaci polyhyperimmune serum. Lane 2 was applied with mAb 1-2 reacted acid, pH 8.0, 0.001 M EDTA, 1 per cent SDS, 5 per cent clonal against Cpsittacistrain A22/Teramo. H High protein molecularweight 13-mercaptoethanol). Protein bands were visualised by standards (in thousands), L Low protein molecular weight standards staining with Coomassie brilliant blue R-250 (Serva), (in thousands) and apparent molecular weights were calibrated by comparison with marker proteins. The SDS-PAGE polypeptide profile of C psittaci isolate A22/Teramo F o r immunocytochemical studies 12 mm diameter showed several protein bands with molecular weights coverslips in 10 cm sealed petri dishes were seeded with McCoy cells in DMEM containing 10 per cent of 35, 42, 52, 58, 62 and 113 k D a (data not shown). Immunoblotting was then performed on electro- FCS. After incubation at 37°C, 5 per cent carbon phoretically separated proteins of partially purified dioxide in water saturated atmosphere until confluchlamydial antigen, according to the methods of ence, monolayers were infected with C psittaci Towbin et al (1979). Transfer to nitrocellulose memA22/Teramo, the sealed petri dishes were centrifuged brane (Schleicher and Schuell) was performed at 400 at 2500 g for 30 minutes, and cycloheximide (1 ktg ml-1) was added to the medium. At 18, 24 and 42 mA for two hours. After transfer, individual 0.5 cm wide strips of the nitrocellulose membrane correspond- hours after infection the coverslips were fixed in absolute methanol for 10 minutes at 37°C, blocked with ing to separated antigen tracks were washed for one hour in PBS, and incubated for 16 to 18 hours either 5 per cent normal goat serum in PBS for 30 minutes, then covered with serial dilutions of either ascitic in serial dilutions of mAbs in PBS or in mouse anti-C psittaci hyperimmune serum. Bound immunoglobulins fluid or mouse anti-C psittaci polyclonal hyperimwere detected by incubating the strips with horseradish mune serum, and incubated for three hours at 37°C in a moist chamber. The immunocytochemical reacperoxidase-conjugated rabbit anti-mouse IgG tion was detected with horseradish peroxidase-con(Dakopatts) diluted 1:200 in PBs containing 10 per cent fetal calf serum (FCS) for 90 minutes at 37°C. The jugated rabbit anti-mouse IgG (Dakopatts) diluted 1:200 in PBS containing 1 per cent normal goat serum strips were developed with a solution of 40 mg and incubated for 30 minutes. The substrate solution 3-3'-diaminobenzidine in 100 ml PBs and 0.1 ml hydrogen peroxide. The molecular weight of the band was was added, and the coverslips incubated for a further 10 minutes, counterstained with haematoxylin, calculated by extrapolation in comparison with marker dehydrated and m o u n t e d in Eukitt (Kindler). proteins stained with amido black. Fig 1 shows that Immunocytochemical staining revealed typical intramAb 1-2 was specific for the 57 kDa antigen.

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Preparation of a monoelonal antibody to Chlamydia psittaci

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animals. Furthermore, mAb 1-2 can be employed as a reagent for antibody epitope mapping on 57 kDa antigen and for defining the molecular structure of chlamydial determinants on 57 kDa antigen. Further studies, using a wider panel of mAbs, are needed to achieve a better understanding of the expression of different immunogenic proteins of C psittaci in the tissues of infected animals. Acknowledgement This research was supported by the National Research Council of Italy. Special project RAISA, subproject number 3, paper number 172. FIG 2: Detection of C psittaci by mAb 1-2 (a) in infected McCoy cells fixed at 42 hours after infection; note the dark staining inclusion bodies, x 4700. (b) In lung sections obtained from experimentally infected mice; the intracytoplasmic inclusion are arrowed, x 1100

cytoplasmic inclusion bodies (Fig 2a) which started to appear 48 hours after infection. Formalin-fixed, paraffin-embedded, 4 pm thick sections obtained from lungs of mice orally infected with C psittaci isolate Bs (Biolatti et al 1991) and from placental tissues of sheep which had experienced chlamydial abortion were deparaffinised with xylol and endogenous peroxidase was blocked with 1 per cent hydrogen peroxide in absolute methanol for 10 minutes and treated as for the coverslips. The sections obtained from lungs of experimentally infected mice showed clearly detectable spherical chlamydial inclusion bodies within the cytoplasm of large cells thought to be macrophages and alveolar epithelial cells, often accompanied by elementary bodies, scattered in the interstitium as a consequence of the rupture of the cytoplasmic membrane of infected cells (Fig 2b). The fetal membranes showed typical inflammatory lesions and characteristic intracytoplasmic inclusions in chorionic epithelial cells (data not shown). Recently, a number of papers have described the importance .of the 57 kDa protein as one of the most important immunogenic proteins associated with chlamydial infections able to invoke a delayed-type hypersensitivity (DTH) response (Morrison et al 1989). DTn reactions to chlamydial antigens appear to be the most important factor in C traehomatis and Cpsittaci infections, and particularly in the pathogenesis of guinea pig inclusion conjunctivitis (Morrison et al 1989). In the course of the pathogenesis of ovine enzootic abortion, DTH reaction plays an important role in protective immunity as shown by the field experience that ewes breed uneventfully in the years subsequent to an ovine enzootic abortion-affected pregnancy (Dawson et al 1986). MAb 1-2 can be used to detect chlamydial antigen in formalin-fixed tissues of experimentally infected

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antibody test with monoclonal antibodies. Microbiology and Immunology 32, 251-263 TOWBIN, H., STACHELIN, T. & GORDON, J. (1979) Eleetrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences USA 76, 4350-4354 TOYOFUKU, H., TAKASHIMA, I., ARIKAWA, J. & HASHI-

MOTO, N. (1986) Monoclonal antibodies against Chlamydia psittaci. Microbiology and Immunology 30, 945-955 WARD, M. E. (1983) Chlamydial classification, development and structure. British Medical Bulletin 39, 109-115

Received June 25, 1991 Accepted July 27, 1992