J. Vet. Med. B 39, 353-361 (1992) 8 1992 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 093 1 - 1793

From the Research Institute for Bacterial Animal Diseases, Jena

Preparation and Characterization of Monoclonal Antibodies against Mycopkzsma bovis EVELYN BERTHOLD, M. HELLER, H. PFUTZNER, R. LEIRERand K. SACHSE" Address of authors: Institut fur Veterinarmedizin des Bundesgesundheitsarntes (Robert von Ostertag-Institut), Bereich Jena, Naumburger Str. 96 a, D-0-6909 Jena 9, Germany

With 6figures and 2 tables (Received for publication November 5, 1991)

Summary Monoclonal antibodies (mabs) against Mycopkzsma (M.) bovis were prepared for use in diagnosis of bovine mastitis. From the original 32 hybridomas actively secreting mabs against M. b o v i , 6 stable lines were cloned. Two of them, Mb 5D8 and Mb 4F6, recognized M. bovis antigens of estimated molecular weights of 33 and 26 kDa, respectively. They showed no cross-reaction to other bovine mycoplasmas, thus rendering them useful for specific detection of this pathogen. All mabs investigated cross-reacted with M . agalactiae which is known to be closely related to M. bovis, but does not occur in cattle. Two other mabs, Mb 5D4 and Mb 1F6, exhibited further cross-reactions to a number of bovine mycoplasma species. Finally, mabs Mb 5D5 and Mb 2G5 reacted with all mycoplasmas tested. The possibility that they recognized constituents of the broth culture medium is discussed.

Introduction Mycoplasma (M.) bowis is considered the most pathogenic bovine mycoplasma

species. It is mainly associated with mastitis, but has also been reported to be involved in arthritis and diseases of the respiratory and genital tracts WASPER, 1982; PFUTZNER, 1984). In the last 15 years, this kind of mastitis also occurred at an increased frequency in Germany (WEIGT, 1980; PFUTZNER,1982). Since there is no effective treatment for mycoplasmal mastitis in the clinical state, control of the spread of the disease in a herd during an epizootic depends on early identification and isolation of infected animals. To reduce the number of new infections a rapid test system for M. bovis detection is necessary. In this paper, we report the production and characterization of monoclonal antibodies (rnabs) against M . bowis for development of a specific antigen ELISA test designed to detect the pathogen directly in milk samples.

Material and Methods Mycoplasma strains and cdtivation All mycoplasma strains investigated (cf. Table 2) were cultivated in medium B (modified Hayflick medium), which supports adequate growth of the majority of Mycopkzsma spp. and

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1983). The field strains were isolated from milk samples of several cattle Acholeplasma spp. (FREUNDT, herds with M. bovis mastitis outbreaks in different countries. Monoclonal antibodies 6- to lo-week-old BAIB/c mice were immunized intraperitoneally with M. bovis strain J 282

whole-cell antigen which consisted of washed cells, 80- to 100-fold concentrated from broth cultures, disrupted by 10 freeze-thaw cycles using liquid nitrogen and 5 x 1 min ultrasonication. For the first immunization, the mice were given 0.5 ml of M. bovis cell suspension emulsified in Freund's complete adjuvant. Two weeks later, the animals were boostered using a cell suspension in Freund's incomplete adjuvant. At least two further injections without adjuvant followed at intervals of two weeks. Four days after the last injection, the mice were killed and spleen cells fused with FO myeloma cells DE ST. GROTHand SCHEIDEGGER, 1980) by standardized techniques (PETERSand BAUMGAR(FAZEKAS TEN, 1990). Hybridomas produced were screened for secretion of M. bovis-specific antibodies by an indirect ELISA. Cultures selected were cloned and used to develop M . bovis-specific antibody tumours in mice (PETERSand BAUMGARTEN, 1990). Antibodies from ascites fluid were recovered by ammonium sulfate precipitation and further purified by affinity chromatography (PETERSand BAUMGARTEN, 1990) using a protein A-Sepharose column (Pharmacia, Freiburg, FRG). Monoclonal antibody isotypes were determined using the Mouse Hybridoma Subtyping Kit (Boehringer, Mannheim, FRG).

SDS-PAGE and immunoblotting Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 10 YO polyacrylamide slab gel was performed according to LAEMMLI(1970). Phosphorylase b (94 kDa), bovine albumine (67 kDa), egg albumine (45 kDa), chymotrypsinogen A (25 kDa), and a-lactalbumine (14.4 kDa), all from Pharmacia, were used as molecular weight markers. Protein transfer to nitrocellulose membranes (SCHLEICHER and SCHUELL,Dassel, FRG) was carried out using the semi-dry mode as described by KYHSE-ANDERSEN (1984). The blots were reversibly stained with Ponceau S (Serva, Heidelberg, FRG) and the positions of standard bands marked. After blocking with 2 % skimmed milk powder solution in 0.01 M phosphate buffered saline, pH 8.3, containing 0.3 YO Tween 20 (PBS/Tween), the nitrocellulose membranes were incubated for 2 h with hybridoma culture supernatant diluted in PBS/Tween (1 :2). Afterwards, they were washed 3 times with PBS/Tween for 20 min, incubated with peroxidase-conjugated protein A (SIFIN, Berlin, FRG) and washed again. Finally, the peroxidase-active bands were visualized with nitro blue tetrazolium chloride (TAKETA,1987). ELISA screening of hybridoma supernatants An ultramicrolitre (UML) format assay was used to screen supernatants and detect possible cross-reactions. The assay, which is carried out in a total volume of 10~1,requires special 96-well UML plates (UML PVC-Blister, Universitat Jena, FRG, Medizin. Fakultat; described by REICHENBACHER et al., 1985). Prior to the ELISA, each well of the UML plates was coated with 10-154 of a 0.5% aminoperoxide solution in chloroform and then incubated with lop1 of antien solution ( 7 ~ g protedml) in coating buffer (0.015M Na2C03, 0.035M NaHC03, 0.2 % NaN,, p H 9.6) at 25 "C overnight. After washing with PBS/Tween, 10 pl culture supernatants diluted 1 : 2 in the same buffer

Table 1. Characterization of monoclonal antibodies to Mycoplasma bovis Hybridoma clone Heavy chain Mb Mb Mb Mb Mb Mb

5D4 1F6 5D8 4F6 5D5 2G5

IgG 2b IgG 1 IgG 2a IgG 2b IgG 1 IgG 1

Antibody isotype Light chain

Antigen recognized in immunoblot (kDa) 33 33 37 26 22-25 22-25

Preparation and Characterization of Monoclonal Antibodies against Mycoplasma bovis


containing 5 % goat serum were added and incubated at 25 "C for 4 h. Alkaline phosphataseconjugated goat anti-mouse immunoglobulin diluted to 1 enzymatic unit per ml served as secondary antibody and was incubated in the same manner. Finally, the substrate 4-methyl umbelliferylphosphate (0.5 mM in 1M diethanolamine, p H 9.6) was added and fluorescence intensity measured after 15-20 min at 365nm using the ELISA processing system SUMAL PE2 (Carl Zeiss Jena, FRG). All samples were run in duplicate. Murine polyclonal M. bowis antiserum served as positive control and mouse preimmune serum as negative control.

Results Mabs secreted by hybridoma clones: About 32 of the original 300 hybridomas actively secreted antibodies against M . bovis whole-cell antigen as determined by the ELISA test. Six stable lines with the highest antibody production were selected for further cloning. Some characteristic properties of these antibodies are given in Table 1. In subtyping, all mabs were found to have light chains of the kappa-isotype, whereas for heavy chains three different isotypes were detected (IgG 1,2a, 2b). Mabs Mb 5D4 and Mb 1F6 both reacted with a 33 kDa band, but differed in heavy chain isotype (IgG 2b and

Table2. Reactivities as determined by immunoblotting and ELISA of mabs Mb 5D4, Mb 5D8, Mb 1F6 and Mb 4F6 with Mycoplasma (M.) and Acholeplasma (A.) spp. including M . bovis reference and field strains Mb 5D4 Mb 5D8 Mb 1F6

M. californicum St-6 M. bovigenitalium PG 11 M.gallinarum PG 16 M. verecundum St- 107 M. arginini G 230 M. canadense 275 C M. bovirhinis PG 43 M. bovoculi M 165f69 M. gatae CS M.alkalescens PG 31fD 12 M. bovine group 7 PG 50 M . mycoides, subsp. mycoides LC type, Y-goat M. agalactiae PG 2 M. gallisepticurn PG 3 1 M.pneumoniae F H M . hyopneumoniue strain J M. hominis PG 21 A . laidlawii PG 8 A. modicum PG 49 A. axanthum S 743 M. bovis Donetta PG 45 M. bovis J 282 M. bovis 365188 M. bovis 937178 M. bovis 221189 M. bovis 589f78 M. bovis 341f84 M. bovis 222f89 M. bowis 422f88 M. bovis 223f89 M. bowis 108f79

Mb 4F6



IgG 1, respectively). The antibodies released by clones Mb 5D5 and Mb 2G5 appear identical by their characteristics and their reaction pattern with a broad band ranging from 22 to 25 kDa. Mycoplasmal antigens recognized by mabs M b liD4, M b liD8, M b 1F6 and M b 4F6: Mycoplasmal proteins recognized by these antibodies were identified by immunoblotting of whole organisms separated by SDS-PAGE. When rabbit hyperimmune serum to M. bovis strain J 282 was used as primary antiserum a large number of reactive bands including those recognized by our mabs were detected (Fig. 1). However, the 26 kDa band of M . agalactiae, which reacted with mab Mb 4F6, was not stained under these conditions (Fig. 1, lane 4). Fig. 2 illustrates that the mabs Mb 5D4, Mb 5D8 and Mb 4F6 reacted with distinct epitopes and that the epitopes recognized were located on discrete macromolecular species. The cross-reacting bands of M . agahctiae, M . bovigenitalium, M . califomicum, M . verecundum and M . gallinarum with Mb 5D4 appeared to be of different molecular weights compared to the corresponding 33 kDa band of M . bovis (Figs. 3a and 3b). For comparison, Fig. 4 shows SDS-PAGE separations of whole-cell proteins of M. bovis and the other cross-reacting Mycophsma species with the protein bands recognized by our mabs. Table 2 presents the reactivities of mabs Mb 5D4, Mb 5D8, Mb 1F6 and Mb 4 F6 with M . bovis type strain Donetta PG 45, strain J 282 and another 9 field strains of this species,

Fig. 1. Immunoblotting analysis of whole cell proteins of various mycoplasmas using polyclonal rabbit antiserum against M . bovis J 282.1 = M.bovis J 282,2 = M.bovigenituliwn, 3 = M . californicum, 4 = M.ugalactiae, 5 = M.arginini. Positions of molecular weight markers are shown on the right (in kDa) The protein bands recognized by the mabs Mb 5D8 (37 kDa), Mb 1F6 and Mb 5D4 (33 kDa) and Mb 4F6 (26 kDa) are indicated on the left of lane 1. Cross-reacting protein bands recognized by Mb 5D4 are indicated on lane 2 and lane 3. O n lane 4, cross-reacting protein bands of Mb 5D8 (37 kDa) and of Mb 1F6 and Mb 5D4 (33 kDa) are indicated

Preparation and Characterization of Monoclonal Antibodies against Mycoplasma bovis


Fig.2. Immunoblotting analysis of M . bovis field strains and M.ugulactiue with mabs Mb 5D4 (directed against 33 kDa antigen), Mb 5D8 (37 kDa) and Mb 4F6 (26 kDa). 1 = M . bovis 221/89, 2 = M . bovis 365/88, 3 = M . bovis 341/84, 4 = M . bovis 422/88, 5 = M.agalactiae. The positions of molecular weight markers are indicated on the right (in kDa)

as well as with 19 strains of important bovine and other animal Mycoplasma and Acholeplasma spp. by immunoblotting and ELISA. All mabs showed cross-reactions with M . agalactiae (see also Fig. 2, lane 5 ) . There were further cross-reactivities of mab Mb 5D4

Fig. 3a. Immunoblotting analysis of mab Mb 5D4. 1 = M . bovis J 282,2 = M . agalactiae, 3 = M . bovigenituliwrn, 4 = M . californicum, 5 = M . gullinarum. Positions of molecular weight markers are given on the right (in kDa)



Fig.3b. Immunoblotting analysis of mab Mb 5D4. 1 = M.bovis J 282, 2 = medium B, 3 = M . bovirhinis, 4 = M . verecundurn, 5 = M . gallinarum. Positions of molecular weight markers are given on the right (in kDa)

Fig.4. Coomassie brillant blue stained whole cell proteins of an SDS-PAGE slab gel (10% w/v acrylamide) of Mycoplasma ( M . ) spp. 1 = M . bovis J 282, 2 = M . agalactiae, 3 = M . bovigenitalium, 4 = M . californicum, 5 = M . verecundurn, 6 = M . gallinarium. The molecular weight markers are indicated on the right (in kDa). The protein bands recognized by the mabs Mb 5D8 (37 kDa), Mb 1F6 and Mb 5D4 (33 kDa) and Mb 4F6 (26 kDa) are indicated on the left of lane 1 and lane 2. The cross-reacting protein bands recognized by Mb 5D4 are indicated on the left of lanes 3-6

Preparation and Characterization of Monoclonal Antibodies against Mycoplasma bowis


Fig. 5. Immunoblotting analysis of mab Mb 5D5 and Mb 2G5. 1 = M . bowis J 282, 2 = ammonium sulfate precipitated horse serum, supernatant, 3 = complete medium B with horse serum, 4 = ammonium sulfate precipitated horse serum, sediment (Ig-fraction, 5 = medium B without horse serum. The positions of molecular weight markers are indicated on the right (in kDa)

with M . bovigenitalium, M . californicum, M . verecundum and M . gallinarum and of Mb 1F6 with M . bovigenitalium. In further test series, strains of Streptococcus (S.) agalactiae, S. dysgalactiae, S. uberis, Staphylococcus aureus and Escherichia coli exhibited no cross-reactions with the 4 mabs considered (results not shown). Mycopkwnal antigens recognized by mabs M b fiDS and M b 2GS: These mabs showed cross-reactions with all mycoplasma species investigated. As can be seen from Fig. 5 , the M. bovis proteins recognized had a molecular weight apparently ranging from 22 to 25 kDa. The mabs also reacted with the uninoculated broth culture medium, with complete horse serum (not shown) and its immunoglobulin fraction (ammonium sulfate precipitate), where broad reactive bands were detected both from 22 to 25 kDa and from 55 to 60 kDa (Fig. 5, lanes 3, 4). Discussion Comparison of whole-cell protein patterns from SDS-PAGE (Fig. 4) indicated the presence of a number of similar antigens in both M.bovis and M. agalactiae. The fact that all mabs cross-reacted with M. agalactiae was not unexpected in view of the close genetic relatedness between the two agents, which formerly had been assigned to the same species. and ERNOinvolving DNA homology and serology led Only in 1976, the studies of ASKAA them to propose their classification as two separate species. However, in practical diagnostics, cross-reactions of mabs between M . agalactiae and M. bovis should be less important because M. agalactiae being strongly adapted to sheep and goat does not occur in cattle. BOOTHBYet al. (1986) used a mab not further specified in M . bovis detection by ELISA, but did not report practical problems due to possible cross-reactions.



The mabs Mb 1F6 and Mb 5D4 recognized the same protein band of M. bovis at 33 kDa and also a 33 kDa band of M . bovzgenitalium. Nevertheless, it is likely that they are binding to distinct epitopes as indicated by the lack of cross-reactions of Mb 1F6 with M. californicum,M. verecundum and M . gallinarum (Table 2) and the different heavy chain isotypes (Table 1). Among the species recognized by mab 5D4 is the group of pathogenic bovine mycoplasmas associated with mastitis, i. e. M . bovis, M. bovigenitalium and M. californicum. This reaction pattern can be used in diagnostic assays to distinguish between mycoplasmal and other forms of mastitis. Generally, cross-reactivities observed were consistent with the inter-species genetic relatedness. WEISBURGet al. (1989) determined subunit rRNA sequences of almost 50 mycoplasma species and their walled relatives as a basis for phylogenetic analysis of these organisms. The authors proposed 5 groups of mycoplasmas. One of them, the “hominis-group” of 16 established species, contained M. cal$ornicum, M . bovigenitalium and M . agalactiue in the same cluster. Unfortunately, the respective sequences of M. bovis, M . gallinarum and M.verecundum were not available from the literature. On the basis of protein band reactivities as revealed by immunostaining it can be derived that despite heat treatment of all antigen samples prior to SDS-PAGE epitopes were still recognized by the mabs, suggesting that they were stable to heat and detergent and that, possibly, the native secondary structure including disulfate bonds was not required for the integrity of the epitope structure. The mabs are obviously directed against epitopes whose binding affinity is neither affected by conformational changes nor determined by tertiary structure. Since whole mycoplasma organisms were used to immunize the mice mabs Mb 5D4, Mb 5D8, Mb 1F6 and Mb 4F6, as expected, recognized major protein antigens of M . bovis which are encountered in all M. bovis strains investigated and also in M . agalactiue (Fig. 4, lanes 1, 2). Major antigens of M . bovis were described in more detail by SACHSEet al. (1992 a). Up to now, only to one of them, the 26 kDa protein recognized by mab Mb 4F6, a biological function has been assigned due to its involvement in the cytadherence process (SACHSEet al., 1992 b). The immunobinding patterns from Fig. 5 suggest mabs Mb 5D5 and Mb 2G5 being directed against the immunoglobulin fraction of equine serum from broth culture medium. Possibly, these components have been integrated into the mycoplasma membrane or at least strongly attached to it. Non-specific protein bands with molecular weights of 22 to 25 kDa and 55 to 60 kDa were also described as serum proteins by YAGUZHINSKAYA (1976). Similarly, MORI et al. (1988) and NICOLET et al. (1980) identified characteristic broad bands encountered in SDS-PAGE patterns and immunoblots of several porcine mycoplasmas as broth medium constituents. Two of the mabs described here, Mb 5D8 and Mb 4F6, were successfully used to develop a specific antigen capture ELISA for detecting M.bovzs in milk samples (HELLER et al., 1992). Acknowledgements The authors gratefully acknowledge the technical assistance of DORIS FABER,RENATEHASS, PEGGY JUGL, UTA MOLLER, ANKER ~ D I G Eand R SANDRATHIERBACH.

Zusammenfassung Monoklonale Antikorper (mAk) gegen Mycopkzsma (M.) bowis, einen bedeutenden Mastitiserreger des Rindes, wurden mit dem Ziel der Anwendung in spezifischen diagnostischen Methoden hergestellr. 6 von ursprunglich 32 Hybridomlinien, die monoklonale Antikorper gegen M. bowis sekretierten, wurden kloniert. Zwei der mAk (Mb 5D8 und Mb 4F6) waren gegen M.bowis-Antigene mit einem Molekulargewicht von ca. 37 bzw. 26 kDa gerichtet. Sie zeigten keine Kreuzreaktionen zu anderen bovinen Mykoplasmen, wodurch sie fur die spezifische Diagnose von Mastitiden bei Kiihen, hervorgerufen durch M.bovis, geeignet sind. Alle untersuchten mAk reagierten mit M.ugutuctiae

Preparation and Characterization of Monoclonal Antibodies against Mycoplusma bovis


einer zu M.bovis eng verwandten, aber an Ziege und Schaf adaptierten Spezies. Bei zwei der hergestellten mAk (Mb 5D4 und Mb 1F6) traten weitere Kreuzreaktionen zu einigen anderen bovinen Mykoplasmenspezies auf. Die mAk Mb 5D5 und Mb 2G5 reagierten mit allen untersuchten Mykoplasmen. Die Moglichkeit, dai3 sie gegen Kulturmedienbestandteile gerichtet sind, wird diskutiert.

References ASKAA,G., and H.ERNo, 1976: Evaluation of Mycoplusmu aguluctiae subsp. bowis to species rank Mycoplasma bovis (HALEet al.) comb. nov. Int. J. Syst. Bacteriol. 26, 323-325. BOOTHBY, J. T., R. MUELLER, D. E. JASPER, and C. B. THOMAS, 1986: Detecting Mycoplusma bovis in milk by enzyme-linked immunosorbent assay using monoclonal antibodies. Amer. J. Vet. Res. 47, 1082-1084. D E ST. GROTH,S., and D. SCHEIDEGGER, 1980: Production of monoclonal antibodies: FAZEKAS strategy and tactics. J. Immunol. Method. 35, 1-24. FREUNDT, E. A., 1983: Culture media for classic mycoplasmas. In: RAZIN,S., and J. G. TULLY(eds.): Methods in mycoplasmology. Val. I, Mycoplasma characterization. Academic Press, New York, 128- 135. HELLER, M., EVELYN BERTHOLD, ANNETTE BRYS,H . PFUTZNER, and R. LEIRER,1992: Antigen capture ELISA tests in ultramicrotitre format using monoclonal antibodies for the detection of Mycoplasma bovzs in milk. Vet. Microbiol. (submitted). JASPER,D.E., 1982: The role of mycoplasma in bovine mastitis. J. Amer. Vet. Med. Assoc. 181, 158-162. KYHSE-ANDERSON, J., 1984: Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J. Biochem. Biophys. Meth. 10, 203-209. LAEMMLI, U.K., 1970: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. MORI, Y., T. HAMAOKA, S. SATO, and S. TAKEUCHI, 1988: Immunoblotting analysis of antibody response in swine experimentally inoculated with Mycoplusmu hyopneumoniue. Vet. Immun. Immunpath. 19,239-250. NICOLET,J., P. PAROZ,and B. KRISTENSEN, 1980: Growth medium constituents contaminating mycoplasma preparations and their role in the study of membrane glycoproteins in porcine mycoplasmas. J. Gen. Microbiol. 119, 17-26. PETERS,J. H., and H . BAUMCARTEN, 1990: Monoklonale Antikorper - Herstellung und Charakterisierung. Springer-Verlag Berlin, Heidelberg, New York, London, Paris, Tokyo, Hong Kong. PFUTZNER, H., 1982: Vorkommen und Bedeutung von Mykoplasmenmastitiden in der DDR. Fachtierarztarbeit Staatsveterinarkunde, Karl-Mam-Univ. Leipzig. H., 1984: Die Mycoplusmu-bovis-Infektion des Rindes. Mh. Vet. Med. 39, 217-220. PFUTZNER, REICHENBACHER, D., G. ULBRICHT,U. GOLKE,J. KILIAN,M. SCHULZEund A. HORN,1985: Zur Frage der visuellen Auswertung eines Ultramikro-ELISA bei Kartoffelviren. Arch. Phytopathol. Pflanzenschutz 21, 95-99. SACHSE,K., C. GRAJETZKI, H. PFUTZNER,and R. HASS, 1992 a: Comparison of Mycoplusmu bovzs strains based on SDS-PAGE and immunoblot protein patterns. J. Vet. Med. B 39, 246-252. SACHSE,K., H. PFUTZNER,M. HELLER,and I. HANEL, 1992 b: Inhibition of Mycoplusmu bowis cytadherence by a monoclonal antibody and various carbohydrate substances. Vet. Microbiol. (submitted). TAKETA, K., 1987: A tetrazolium method for peroxidase staining. Application to the antibody-affinity blotting of a-fetoprotein separated by lectin affinity electrophoresis. Electrophoresis 8, 409 -41 4. WEIGT,U., 1980: Mycoplusmu bowis als Mastitis- und Arthritiserreger in einem Rinderbestand (Vorlaufige Mitteilung). Berl. Munch. Tierarztl. Wschr. 93, 206. WEISBURG, W.G., J.G. TULLY, D.L. ROSE, J.P. PETZEL,H.OYAIZU,D.YANG, L.MANDELO, T. G. LAWRENCE, J. VAN ETTEN,J. MANILOFF,and C. R. WOESE,1989: A phyJ. SECHREST, logenetic analysis of the mycoplasmas basis for their classification. J. Bacteriol. 171, 6455-6467. YAGUZHINSKAYA, 0.E., 1976: Detection of serum proteins in the electrophoretic patterns of total proteins of mycoplasma cells. J. hyg. 77, 184-198.

Preparation and characterization of monoclonal antibodies against Mycoplasma bovis.

Monoclonal antibodies (mabs) against Mycoplasma (M.) bovis were prepared for use in diagnosis of bovine mastitis. From the original 32 hybridomas acti...
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