Veterinary Microbiology, 30 ( 1992 ) 3 5 - 4 6 Elsevier Science Publishers B.V., A m s t e r d a m
35
A monoclonal blocking ELISA detecting serum antibodies to Mycoplasma hyopneumoniae Niels C. Feld, Per Qvist, Peter Ahrens, Niels F. Friis, and Anders Meyling National Veterinary Laboratory, P.O. Box 373, DK-1503 Copenhagen V, Denmark (Accepted 14 June 1991 )
ABSTRACT Feld, N.C., Qvist, P., Ahrens, P., Friis, N.F. and Meyling, A., 1992. A monoclonal blocking ELISA detecting serum antibodies to Mycoplasma hyopneumoniae. ~Let.MicrobioL, 30: 35-46. A monoclonal blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies
to Mycoplasma hyopneumoniae in porcine serum has been developed. The monoclonal antibody (mAb) reacts with an M. hyopneumoniae specific epitope on a molecule of approximately 74 kDa. Only sera from M. hyopneumoniae infected pigs were able to block the binding of the mAb although antibodies from M. flocculare infected pigs also recognized a 74 kDa molecule. Sera from experimentally infected pigs as well as field samples were compared by the ELISA and by an indirect hemagglutination assay (IHA). In experimental pigs, the earliest detectable antibody response was found to be almost identical for both assays, but for some of the pigs the time of detection was significantly earlier by blocking ELISA than by IHA. In naturally infected herds more samples were found to be positive by ELISA than by IHA. Furthermore, the results indicate that sera from naturally M. flocculare infected pigs may give rise to cross-reactions in the IHA. The blocking ELISA appears to be a valuable and repr Oducible tool in the surveillance and serodiagnosis of M. hyopneumoniae infections in pigs.
INTRODUCTION
A specific and sensitive serological test for detection of antibodies to Mycoplasma hyopneumoniae in porcine serum is required for surveillance and diagnosis of M. hyopneumoniae infections in specific pathogen free (SPF) pig herds. Over the years a number of different methods have been developed including complement fixation assay (CF) (Boulanger and L'Ecuyer, 1968 ), indirect hemagglutination assay (IHA) (Goodwin et al., 1969 ), and enzymelinked immunosorbent assay (ELISA) (Bruggmann and Keller, 1977), but none of these techniques are entirely satisfactory. The indirect ELISAs (Bruggmann and Keller, 1977; Nicolet et al., 1980) described so far are sensitive, but are based on the use of crude antigen preparations. This means that cross reactions brought about by the commonly occurring, and closely related M. flocculare are possible. Blocking or competitive ELISAs for antibody determination may be de0 3 7 8 - 1 1 3 5 / 9 2 / $ 0 5 . 0 0 © 1992 Elsevier Science Publishers B.V. All rights reserved.
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N.C. FELD ETAL.
signed in such a way that the specificity of the assay is determined by the detector antibody rather than by the antigen. By use of a monoclonal antibody (mAb) in a blocking ELISA it is possible to achieve specificity at the epitope level, even when crude antigen is used. In this report the development of a blocking ELISA for the detection of antibodies to M. hyopneumoniae in porcine sera and the mAb used as detector antibody is described. MATERIALS AND METHODS
Antigen production. The type strain "J" of M. hyopneumoniae was used for production of antigen for ELISA and for raising polyclonal antibodies in rabbits. For ELISA and for immunization of mice the mycoplasmas were grown in the m e d i u m of Hovind-Hougen and Friis ( 1991 ) modified as follows: Pig serum was omitted and horse serum used at 20%. A 10% sterile glucose solution was added to 0.2% and pH adjusted to 7.6. The mycoplasmas were harvested late in the stationary growth phase by centrifugation at approx. 15 000 g for 1 h and then washed three times in 0.9% NaC1 solution. Crude antigen was prepared by resuspending the cell-pellet in phosphate buffered saline (PBS; 10 m M phosphate, 0.15 M NaC1, pH 7.2) to 1 m g / m l followed by freezing. To prepare antigen for the monoclonal blocking ELISA, the cell-pellet was resuspended in 18 ml water and sonicated on ice for 45 sec. (100 W, 20 KC). Insoluble material was subsequently removed by centrifugation at 10 000 g for 15 min.
Rabbit antiserum. Polyclonal antibodies to M. hyopneumoniae were produced by immunization of rabbits with antigen harvested from rabbit broth cultures as described previously (Friis, 1977; Friis and Jensen, 1984). Serum from several bleedings was pooled and fractionated by a m m o n i u m sulphate precipitation (Harboe and Ingild, 1983 ), and the globulin fraction was used for coating the ELISA plates (see below).
Experimental infection of pigs. Reference sera to M. hyopneumoniae were prepared in 6 M. hyopneumoniae free, 12 week-old SPF pigs. Three of the pigs were infected once by aerosol inhalation for 7 min with 5 × 108 color changing units (ccu) of three pooled field strains of M. hyopneumoniae which had been filtered through a 0.45/~m filter and cloned once from solid medium. The other three pigs were contact infected by their penmates. All pigs were bled on the day of infection and then at weekly intervals for 3 months. To investigate cross reactions between M. hyopneumoniae and M. flocculore three caesarian section-derived, colostrum-deprived, 3 week-old piglets kept in isolation were infected (aerosol a n d / o r nasal instillation on days 0, 6,
MONOCLONAL BLOCKING ELISA TO MYCOPLASMA IIYOPNEU3,1ONIAE
37
35 and 41 ) with 107 ccu of a pool of two filter-cloned field strains of M.
flocculare. Production of monoclonal antibodies. Female BALB/c mice (8-12 weeks) were immunized subcutaneously three times three weeks apart with 200/tl of an equal mixture of M. hyopneumoniae crude antigen and Freund's incomplete adjuvant. The mice were boosted intravenously with 200/tl crude antigen three days before fusion. Spleen cells from immunized mice were fused with myeloma P3-X63-Ag8.653 cells (Kearny et al., 1979) using 50% (w/v) polyethylene glycol 4000 as described by Galfre and Milstein ( 1981 ), except that human endothelial culture supernatant (Costar, Badhoevedorp, Netherlands) was used instead of feeder cells (Astaldi et al., 1980). Growing hybridomas were screened by a competitive ELISA and cloned twice by limiting dilution. Briefly, competitive ELISA was performed essentially as described for blocking ELISA (see below), except that crude antigen was used for plate coating and mAb was demonstrated by incubation with horseradish peroxidase ( H P R ) conjugated rabbit anti-mouse immunoglobulin (Dako, Glostrup, Denmark). The binding of immunoglobulin from hybridoma culture supernatant (HCS) to antigen was inhibited by introducing a pool of sera from the pigs experimentally infected with M. hyopneumoniae as an intermediate layer. HCSs giving an absorbance, Apre, of more than 1.0 absorbance unit with pre-infection sera as the intermediate layer and less than 50% of Aore when the antibody-positive sera was used, were considered positive. Isotype and light chain of the mAb was determined in HCS by immunoblot assay as described by McDougal et al. (1983) using rabbit anti-isotype antisera and rabbit anti-light-chain antisera (Miles Scientific, Naperville, Illinois). The mAb was purified from HCS by affinity chromatography, using protein-A immobilized on agarose (Kem-En-Tec, Copenhagen, Denmark), and biotinylated according to the procedure described by Guesdon et al. ( 1979 ).
Immunoblotting. Crude antigen was submitted to electrophoresis according to standard procedures (Laemmli, 1970). The separated molecules were transferred to a 0.45/~m nitrocellulose membrane (Whatman, Maidstone, England) using a semidry electroblotter (Kem-En-Tec, Copenhagen, Denmark) as described by Kyhse-Andersen (1984). The transferred molecules were visualized either by staining with collodial gold (Moremans et al., 1985 ) or by immunological development essentially as described by Bjerrum et al. (1987). The molecular mass was estimated by comparison with known marker proteins: Ovalbumin (43.0 kDa), BSA (66.3 kDa), polymerase fl (150.6 kDa) and fl' ( 155.2 kDa) and myosin (approx. 200 kDa ) (Nene and Glass, 1984 ).
Dot blot assay. The species specificity of the mAb was investigated by a dot
38
N.C. F E L D ET AL.
blot assay using 20 field strains of M. hyopneumoniae, nine field strains plus one type strain (Ms42) of M. flocculare, seven field strains of M. hyorhinis, two field strains ofM. hyosynoviae, and one field strain ofM. dispar. Briefly, 105, 104 and 103 ccu of each strain was applied in a volume of 2 #1 to a nitrocellulose membrane and, after blocking with PBS containing 2% Tween 20 (PBS-T), the membrane was incubated for 1 h with a 1 : 10 dilution of HCS. Bound mAb was demonstrated as described for immunoblotting.
Blocking ELISA. The blocking ELISA was performed as follows: 96 well microtiter plates (Nunc, Roskilde, Denmark) were coated with 100/tl per well (all subsequent volumes are 100/~1 per well ) of rabbit immunoglobulin to M. hyopneumoniae diluted 1 : 1000 in 0.1 M carbonate buffer, pH 9.6. After incubation overnight at 4 ° C the plates were tapped dry and blocked for 1 h with PBS containing 4% (w/v)skim milk (PBS-M). The plates were washed in four changes of PBS-T followed by incubation for 1 h at room temperature with M. hyopneumoniae antigen diluted 1 : 250 in PBS-T. After another wash, serum samples diluted 1 in 10 in PBS-M were added in duplicate and the plates incubated for 2 h. PBS-M alone was applied to one row and both positive and negative control serum was added to four wells each. Without emptying, biotinylated mAb 17 diluted 1 : 2000 in PBS-M was added, and the plates were incubated for 15 min at room temperature. The plates were washed and incubated for 1 h with HRP-conjugated avidin (Dako, Glostrup, Denmark) diluted 1 : 8000 in PBS-T. After a final wash, enzyme substrate (8 mg 1,2orthophenyldiamine dihydrocloride, 12 ml 0.1 M citrate, pH 5 and 5 #1 H202 ) was added, and after 20 min colour development was stopped with 0.5 M H 2 S O 4. The spectrophotometric absorption was read at 490 nm using 650 nm as reference. The absorption mean of the row without pig serum was used for calculation of the percent inhibition by individual sera. The dilution and incubation time for biotinylated mAb 17 and pig serum were adjusted to give 0-20% inhibition with negative sera and 95-98% inhibition with sera from the pigs experimentally infected with M. hyopneumoniae. Preliminary 600 negative serum samples were investigated for confirming the adjustment. A test result was considered positive for inhibition >/50%.
IHA method. Fixed and tanned red blood cells (RBC) were sensitized with an extract of soluble antigens essentially as described by Holmgren (1974) with the modification that the RBC were fixed in pyruvic aldehyde (Rust et al., 1972 ). Complete agglutination of the RBC in serum dilution 1 : 80 or above was considered positive.
Field sera. Pig sera from SPF herds and SPF herds infected with M. hyopneumoniae were examined by blocking ELISA and by IHA. Group A consisted of 1000 sera obtained from 17 SPF herds without clinical signs of M. hyopneu-
MONOCLONAL BLOCKING ELISA TO MYCOPL,ISMA ltYOPNEUMONIAE
39
moniae infection but which occasionally had had a few reactors by IHA. Group B consisted of 498 serum samples from 46 SPF herds probably free of M. hyopneumoniae infection. Group C consisted of 462 samples from 23 SPF herds infected with M. hyopneumoniae and from which M. hyopneumoniae has been isolated. RESULTS
Monoclonal antibodies. A large number of hybridomas were identified but only supernatant from a single clone appeared positive in the competitive ELISA. This mAb, belonging to the IgG 1 t¢ isotype, was designated mAb 17. The molecular specificity of mAb 17 was demonstrated by immunoblotting (Fig. 1 ). When crude M.
A 1 2 3 4 5
B 1 2 3 4 5
C 1234
D 5
1
190 -122 - 94~ 74-64-49 4 4 --c 37--
Fig. I. Immunoblot analysis ofM. hyopneumoniae antigens using monoclonal antibody and sera collected sequentially from pigs infected with M. hyopneumoniae and M. flocculare. (A) Serum ( 1 : 100) from a pig experimentally infected with M. hyopneumoniae. Preinfection serum (A1) and serum obtained 2 weeks post infection (wpi) (A2), 4 wpi (A3), 6 wpi (A4) and 8 wpi (A5). (B) Serum ( 1 : 100) from a pig in contact with the pig in A above. Preinfection serum (B1) and serum obtained 2 wpi (B2), 4 wpi (B3), 6 wpi (B4) and 8 wpi (B5). (C) Serum ( 1 : 100) from a pig infected with M. flocculare. Preinfection serum (C 1) and serum obtained 2 wpi (C2), 4 wpi (C3), 6 wpi (C4) and 13 wpi (C5). (D) Hybridoma culture supernatant, mAb 17 ( 1 : 100), (D1).
40
N.C. F E L D E T A L .
hyopneumoniae antigen was used, mAb 17 recognized a molecule of approx. 74 kDa (Fig. 1, lane DI ). Four weeks after experimental infection with M.
hyopneumoniae, pig serum antibodies to molecules of approx. 122, 94, 74, 49 and 44 kDa were identified on immunoblots (Fig. 1, lane A3). After 8 weeks additional bands at approx. 190, 98, 93, 85, 80, 64, 52, 42 and 37 kDa were seen (Fig. 1, lane A5 ). The same pattern, although delayed by two weeks, was observed with serum from a pig kept in contact with the experimentally infected ones (Fig. 1), lanes B1-B5). Preimmune sera from the animals had antibodies to the 49 kDa molecule (Fig. l, lane A1 ) or both the 74 and 49 kDa molecules (Fig. 1, lane B1 ). These two patterns were also found in the other four pigs included in this experiment (data not shown). Eight weeks after infection with M. flocculare, antibodies to the 49 and 44 kDa molecules of M. hyopneumoniae were detected on immunoblots (Fig. 1, lane C4) and after 13 weeks antibodies to the 74 kDa molecule were also identified (Fig. 1, lane C5 ). Preinfection serum from this animal was negative by immunoblotting (Fig. 1, lane C 1 ). One of the two remaining caesarian section derived pigs confirmed this while preinfection serum from the last pig had antibodies to the 49 kDa molecules (data not shown).
Strain specificity ofmAb 17. The reactivity of mAb17 with other species of mycoplasma was investigated by dot blot assay, mAb 17 was shown to react with 103 ccu for all of the 20 field strains ofM. hyopneumoniae investigated. No reactions of mAb 17 were seen with the 105 ccu from any strain of M. flocculare, M. hyorhinis, M. hyosynoviae and M. dispar.
ELISA adjustment. In a preliminary study the mean inhibition percentage of 600 negative serum samples investigated over several days was 13.7 with a standard deviation of 10.3%. For the same investigation the inhibition of an internal positive standard was 70.7_+ 2.7%. The analytical sensitivity of the ELISA is mainly determined by the concentration and incubation time for the mAb but even a 10 min increase of incubation time only reduced the inhibition of the internal standard by 2.7%.
Experimentally infected pigs. All animals experimentally infected with M. hyopneumoniae showed a positive antibody response but at different intervals after infection. By ELISA the experimentally infected pigs showed a positive antibody response after two weeks. The titers peaked 8-9 weeks post infection and showed a slight decrease toward the end of the observation period. Contact infected pigs showed positive antibody response after 4-5 weeks followed by a rapid increase in titer so that the peak value and decline were mostly consistent with the values for experimentally infected pigs. A detectable antibody response was observed by IHA in the experimental pigs 2-5 weeks
M O N O C L O N A L B L O C K I N G ELISA T O
MYCOPLASMA HYOPNE[~,IONIAE
41
100"
.E
80"
_tJ
60"
//" /
/ /i II =-
20
g
/--'
t
"~
.,SJ
'"
0
1'o
o
1'2
1'4
weeks pest infection Fig. 2. Inhibition by sequential serum samples, diluted 1: 11, from pigs experimentally infected ( ) and contact infected (---) with M. hyopneumoniae.Day zero is the day of infection. A test result was considered positive for inhibition >/50%. after infection. For the contact infected pigs a positive antibody response was detected after 4-6 weeks. The m a x i m u m titer 1:2560 for experimentally infected pigs was identical in both assays. By IHA, for one pig the m a x i m u m titer was 80, while another showed the first detectable antibody response six weeks post infection. Serum from these pigs did not differ significantly by blocking ELISA from the other experimental pigs (Fig. 2 ). Three M. flocculare infected pigs having a homologous IHA titer i> 160 after 11 weeks showed no inhibition in the blocking ELISA during the investigation period.
Field samples. In group A serum samples, all were found to be negative in the blocking ELISA. One of the 17 herds had several reactors by IHA with titers >1640. M. flocculare was isolated from this herd. In group B one herd was found to be positive by both assays with eight reactors in ELISA and five reactors in IHA. This result was confirmed by isolating M. hyopneumoniae. The remaining herds were negative by ELISA but by IHA a single reactor was found in each of six herds. In the following three months no clinical sign of infection was noticed in these herds. In group C (Table 1 ) ELISA and IHA reactors were found in all except two herds which were negative by IHA. All samples positive by IHA were also positive by ELISA. In this group of chronically infected herds a total of 73 per cent of the samples tested positive by blocking ELISA compared with only 30 per cent by IHA. The ELISA inhibition m e a n for serum samples with IHA titer 80, 160 and >1320 was 84 + 12%, 92 _+8% and 94 ___3%, respectively.
42
N.C. FELD ET AL.
TABLE 1 Comparison o f blocking ELISA and IHA using sera from M. hyopneumoniae infected herds. Herd No.
No. tested ~
ELISA pos. 2
Mean inhibition ELISA pos. (S.D.)3
no. (%) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 TOTAL
18 20* 20* 20 21" 20 20* 30* 20* 20 20 19" 19" 21 20* 18"* 20 20* 19 19" 19 19" 20 462
12 18 11 16 17 11 15 23 19 14 15 17 9 14 19 10 9 16 16 16 15 14 9 335
(67) (90) (55) (80) (81) (55) (75) (77) (95) (70) (75) (89) (47) (67) (95) (56) (45) (80) (84) (84) (79) (74) (45) (73)
78.8 73.7 71.5 88.3 74.7 72.5 77.8 83.7 83.5 71.4 75.5 91.2 70.0 83.8 93.8 66.9 77.9 82.8 83.4 77.5 74.1 76.7 70.9
(17.5) (12.9) (13.3) (9.3) (11.4) (15.2) (11.2) (8.9) (14.0) (13.7) (13.4) (4.6) (12.8) (14.1) (5.8) (12.0) (16.7) (17.1) (12.1) (13.0) (16.9) (14.1) (15.2)
IHA
pos. 4
Distribution o f 1HA titer 5
no. (%)
80
160
>/320
7 (39) 9 (47) 3 (16) 11 (55) 6(30) 3(15) 5 (26) 12 (41) 13 (68) 2 (10) 3(15) 3 (17) (0) 6 (29) 11 (58) (0) 7 (35) 11 (58) 5 (26) 3 (17) 1 (5) 7 (39) 6 (30) 132 (30)
4 5 2 2 5 1 4 10 3 2 1 2
2 3 1 1 1 1 2 1 1
3 2
2 3
5 3 4 3 4 4
3 1 2 1
1 l 8 I 1 2 8 1 1 6 2 4 1 1 1
*One sample not testable by IHA due to spontaneous hemagglutination. ~The n u m b e r o f serum samples tested in each herd. 2The n u m b e r o f reactors found by ELISA and (percent). 3The ELISA mean inhibition of positive samples for each herd and the standard deviation in parenthesis. 4The n u m b e r o f reactors found by IHA and (percent). SThe IHA reactors distributed by titer.
DISCUSSION
The aim of the present project was to develop a specific and sensitive serological ELISA based on monoclonal antibody competing with natural antibodies to M. hyopneumoniae in porcine serum, mAb 17 appeared to be the first monoclonal antibody with this property to be described for M. hyopneumomiae immunoassays. It was demonstrated that mAb 17 reacted with a molecule banding at approx. 74 kDa after SDS-PAGE and immunoblotting. Mori et al. ( 1988 ) demonstrated antibodies to the 74 kDa molecule in por-
MONOCLONAL BLOCKING ELISA TO MYCOPLASMA HYOPNEL~VIONIAE
43
c~ne hy-perimmune serum to M. hyopneumoniae, M. flocculare and M. hyor~hinis. Others found molecules of approx, this mass in all three mycoplasmas (B/51ske et al., 1987). This reactivity appears to involve epitopes other than the one recognized by mAb 17 because only porcine antiserum to M. hyopneumoniae was able to inhibit the binding of mAb 17 in competitive ELISA. The experimental pigs were from a certified herd and hardly sensitized to M. hyopneumoniae before the experimental exposure. The positive reaction in immunoblot of the preinoculation sera could represent cross-reactions with other microorganisms. Recently, it was reported that the gene coding for a 74 kDa protein shared sequence similarity with DnaK ofEscherichia coli (Birkel a n d et al., 1990). Sequences from this protein, one of the family of highly conserved 70 kDa heat shock proteins, seems to be expressed by a variety of microorganisms (Topp and Faulds, 1990), and this could give rise to the reactions observed by immunoblotting. Most of the cae arian section derived, colostrum deprived pigs were probably not colonized by a large number of different microorganisms and therefore were negative in immunoblotting. Nicolet et al. (1980) found that the use of ultrasonically disintegrated M. hyopneumoniae antigen in an ELISA caused non-specific binding of porcine immunoglobulins and Mori et al. (1987) observed non-specific binding of mAbs (especially IgG isotypes) to porcine immunoglobulins. Our prelimi.nary results confirm these findings but by the use of rabbit immunoglobulin as the catching antibody in the ELISA, non-specific binding of mAb 17 (IgG1 to) was eliminated. Sera from experimentally infected pigs and from herds with known M. hyopneumoniae status were compared by IHA and ELISA for evaluation of the blocking ELISA. Using the calculated inhibition percentage for experimental animals, a clear distinction between inoculated and contact infected animals was observed (Fig. 2 ). Using the ELISA, experimental infected pigs were found to have seroconverted between the first and the second week. This time of seroconversion is among the earliest reported in literature for M. hyopneumoniae immunoassays (Lloyd et al., 1987). In contrast to Armstrong et al. (1983) we found no difference in titer between experimental infected and contact infected pigs. A gradual decrease in titer observed in experimental pigs after week 9 (results not shown ) was hardly visible from the inhibition curve (Fig. 2 ). Due to the difficulty of isolating M. hyopneumoniae, the assays were compared using test-samples from SPF-herds infected with M. hyopneumoniae. This comparison showed a significantly increased number of reactors for the ELISA (Table 1 ). Neither the weak, nor the slow IHA responding pig were significantly different from the other experimental pigs when examined by ELISA. The negative IHA result observed in two of the herds in group C may be due to the time of seroconversion detected by this assay. In chronically infected herds, piglets are protected by maternal antibodies for the first cou-
44
N.C. FELD ET AL.
ple of months and antibodies due to infection are first detected at 90-150 days of age (Mori et ah, 1987). The time of seroconversion is dependent on farm management, infection pressure and the assay used. Test-samples in this investigation were from 3-5 month old pigs. The specificity of M. hyopneumoniae immunoassays is often evaluated with sera from pigs experimentally infected with other mycoplasmas. In agreement with Armstrong et al. ( 1987 ) we found that pigs experimentally infected with M. flocculare were slow to seroconvert and when they did, titers were low and antibodies to M. hyopneumoniae were not detectable. In contrast, we found by IHA some naturally M. flocculare-infected herds (M. flocculare isolated) with high-titered cross-reacting antisera to M. hyopneumoniae. The herds with few reactors by IHA (group A and B) were intensively surveyed at the slaughter line and by clinical inspection in the herds but no history ofM. hyopneumoniae infection could be verified. As quoted by others (Armstrong et al., 1983) IHA is not an ideal assay for surveillance ofM. hyopneumoniae infection because ofunspecificity and difficulty in the reproducibility of alike antigen coated RBC. The described blocking ELISA may prove to be an advantage in the serodiagnostic and surveillance of M. hyopneumoniae infections in pigs. ACKNOWLEDGEMENTS
We thank DVM Kristen Barfod and DVM Vibeke Sorensen, The Federation of Danish Pig Producers and Slaughterhouses (Vet. Dept.), Roskilde, Denmark, for kindly supplying us with information and sera from SPF pig herds. This work was supported in part by the Danish Agricultural and Veterinary Research Council.
REFERENCES Ai~mstrong, C.H., Freeman, M.J., Sands-Freeman, L., Lopez-O-suna, M., Young, T. and Runnels, L.J., 1983. Comparison of the enzyme-linked immunosorbent assay and the indirect hemagglutination and complement fixation test for detecting antibodies to Mycoplasma hyopneumonia. Can. J. Comp. Med., 47: 464-470. Armstrong, C.H., Sands-Freeman, L. and Freeman, M.J., 1987. Serological, pathological and cultural evaluations of swine infected experimentally with Mycoplasrna flocculare. Can. J. Vet. Res., 51: 185-188. Astaldi, G.C.B., Janssen, M.C., Lansdorp, P., Wiellems, C., Zeijlemaker, W.P. and Oosterhof, F., 1980. Human endothelial culture supernatants (hecs): A growth factor for hybridomas. J. Immunol., 125:1411-1414. Birkelund, S., Lundemose, A.G. and Christiansen, G., 1990. The 75-Kilodalton cytoplasmic Chlamydia trachomatis L2 polypeptide Is a DnaK-like protein. Infect. Immun., 58: 20982104. Bjerrum, O.J., Selmer, J.C. and Lihme, A., 1987. Native immunoblotting: Transfer of membrane proteins in the presence of non-ionic detergent. Electrophoresis, 8: 388-397.
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B61ske, G., Strandberg, M., Bergstr6m, K. Johansson, K., 1987. Species-specific antigens of Mycoplasma hyopneumoniae and cross-reactions with other porcine mycoplasmas. Curr~ Microbiol. 15: 233-239. Boulanger, P. and L'Ecuyer, C., 1968. Enzootic pneumoniae of pigs: Complement-fixation tesi for the detection of mycoplasma antibodies in the serum of immunized rabbits and infected' swine. Can. J. Comp. Med., 32: 547-554. Bruggmann, S., and Keller, H., 1977. Quantitative detection of antibodies to Mycoplasma suipneumoniae in pigs' sera by an enzyme-linked immunosorbent assay. Vet. Rec., 101: 109111. Friis, N.F., 1977. Mycoplasma suipneumoniae and Mycoplasmaflocculare in the growth precip, itation test. Acta Vet. Scand., 18:168-175. Friis, N.F., and Jensen, P.T., 1984. Serological comparison of type strains of porcine, bovine; and ovine mycoplasmas with atypical colony morphology. Acta Vet. Scand., 25: 29-35. Galfrb, G., and Milstein, C., 1981. Preparation of monoclonal antibodies: Strategies and pro-' cedures. Methods. Enzymol., 73: 1-46. Goodwin, R.F.W., Hodgson, R.G., Whittlestone, P. and Woodhams, R.L., 1969. Immunity in experimentally induced enzootic pneumoniae of pigs. J. Hyg. Camb., 67:193-207. Guesdon, J.-L., Ternynck, T. and Avrameas, S., 1979. The use of avidin-biotin interaction in, immunoenzymatic techniques. J. Histochem. Cytochem., 27:1131-1139. Harboe, N.M.G. and Ingild, A., 1983. Immunization, isolation of immunoglobulins and antibody titre determination. Scand. J. lmmunol., 17:345-351. Holmgren, N., 1974. An indirect hemagglutination test for detection of antibodies against Mycoplasma hyopneumoniae using formalized tanned swine erythrocytes. Res. Vet. Sci., 16: 341-346. Hovind-Hougen, K. and Friis, N.F., 1991. Morphological and ultrastructural studies of Mycoplasrnaflocculare and Mycoplasma hyopneumoniae in vitro. Res. Vet. Sci., in press. Kearny, J.F., Radbruch, A., Liesegang, B. and Rajewsky, K., 1979. A new mouse myeloma cell line that lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J. Immunol., 123:1548-1550. Kyhse-Andersen, J. 1984. Electroblotting of multiple gels: A simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J. Biophys. Biochem. Methods, 10: 203-209. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly c~fthe head of bacteriophage T4. Nature (London), 227: 680-685. Lloyd, L.C., Cottew, G.S. and Anderson, D.A., 1987. Early serological responses to Mycoplasma hyopneumoniae infection. Israel J. Med. Sci., 23: 647-649. McDougal, J.C., Browning, S.W., Kennedy, S. and Moore, D.D., 1983. lmmunodot assay fordeterming the isotype and light chain of murine monoclonal antibodies in unconcentrated hybridoma culture supernatants. J. Immunol. Methods, 63:281-290. Moremans, M., Daneels, G. and De Mey, J., 1985. Sensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes. Anal. Biochem., 145:315-321. Mori, Y., Hamaoka, T. and Sato, S., 1987. Use of monoclonal antibody in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Mycoplasma hyopneumoniae. Israel J. Med. Sci., 23: 657-662. Mori, Y., Hamaoka, T., Sato, S. and Takeuch, S., 1988. Immunoblotting analysis of antibody response in swine experimentally inoculated with Mycoplasma hyopneumoniae. Vet. Immunol. Immunopathol., 19: 239-250. Nene, V. and Glass, R.E., 1984. Genetic studies on the fl subunit ofEscherichia coli RNA polymerase. Mol. Gen. Genet., 194:166-172.
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Nicolet, J., Paroz, P. and Bruggmann, S., 1980. Tween 20 soluble proteins ofMycoplasma hyopneumoniae as antigen for an enzyme-linked immunosorbent assay. Res. Vet. Sci., 29: 305309. Rust, J.H., Berman, S., Habig, W.H., Marshall, J.D. and Cavanaugh, D.C., 1972. Stable reagent for the detection of antibody to the specific fraction 1 antigen of Yersinia pestis. Appl. Microbiol., 93: 721-724. Topp, C., and Faulds, D.H., 1990. Cloning and nucleotide sequence of the gene for the 74.5 kDa antigen, and HspT0 homologue in Mycoplasma hyopneumoniae. Abstr. 8th Int. Congr. IOM, Istanbul, Turkey, IOM lett., 1: 270-271.
35
A monoclonal blocking ELISA detecting serum antibodies to Mycoplasma hyopneumoniae Niels C. Feld, Per Qvist, Peter Ahrens, Niels F. Friis, and Anders Meyling National Veterinary Laboratory, P.O. Box 373, DK-1503 Copenhagen V, Denmark (Accepted 14 June 1991 )
ABSTRACT Feld, N.C., Qvist, P., Ahrens, P., Friis, N.F. and Meyling, A., 1992. A monoclonal blocking ELISA detecting serum antibodies to Mycoplasma hyopneumoniae. ~Let.MicrobioL, 30: 35-46. A monoclonal blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies
to Mycoplasma hyopneumoniae in porcine serum has been developed. The monoclonal antibody (mAb) reacts with an M. hyopneumoniae specific epitope on a molecule of approximately 74 kDa. Only sera from M. hyopneumoniae infected pigs were able to block the binding of the mAb although antibodies from M. flocculare infected pigs also recognized a 74 kDa molecule. Sera from experimentally infected pigs as well as field samples were compared by the ELISA and by an indirect hemagglutination assay (IHA). In experimental pigs, the earliest detectable antibody response was found to be almost identical for both assays, but for some of the pigs the time of detection was significantly earlier by blocking ELISA than by IHA. In naturally infected herds more samples were found to be positive by ELISA than by IHA. Furthermore, the results indicate that sera from naturally M. flocculare infected pigs may give rise to cross-reactions in the IHA. The blocking ELISA appears to be a valuable and repr Oducible tool in the surveillance and serodiagnosis of M. hyopneumoniae infections in pigs.
INTRODUCTION
A specific and sensitive serological test for detection of antibodies to Mycoplasma hyopneumoniae in porcine serum is required for surveillance and diagnosis of M. hyopneumoniae infections in specific pathogen free (SPF) pig herds. Over the years a number of different methods have been developed including complement fixation assay (CF) (Boulanger and L'Ecuyer, 1968 ), indirect hemagglutination assay (IHA) (Goodwin et al., 1969 ), and enzymelinked immunosorbent assay (ELISA) (Bruggmann and Keller, 1977), but none of these techniques are entirely satisfactory. The indirect ELISAs (Bruggmann and Keller, 1977; Nicolet et al., 1980) described so far are sensitive, but are based on the use of crude antigen preparations. This means that cross reactions brought about by the commonly occurring, and closely related M. flocculare are possible. Blocking or competitive ELISAs for antibody determination may be de0 3 7 8 - 1 1 3 5 / 9 2 / $ 0 5 . 0 0 © 1992 Elsevier Science Publishers B.V. All rights reserved.
36
N.C. FELD ETAL.
signed in such a way that the specificity of the assay is determined by the detector antibody rather than by the antigen. By use of a monoclonal antibody (mAb) in a blocking ELISA it is possible to achieve specificity at the epitope level, even when crude antigen is used. In this report the development of a blocking ELISA for the detection of antibodies to M. hyopneumoniae in porcine sera and the mAb used as detector antibody is described. MATERIALS AND METHODS
Antigen production. The type strain "J" of M. hyopneumoniae was used for production of antigen for ELISA and for raising polyclonal antibodies in rabbits. For ELISA and for immunization of mice the mycoplasmas were grown in the m e d i u m of Hovind-Hougen and Friis ( 1991 ) modified as follows: Pig serum was omitted and horse serum used at 20%. A 10% sterile glucose solution was added to 0.2% and pH adjusted to 7.6. The mycoplasmas were harvested late in the stationary growth phase by centrifugation at approx. 15 000 g for 1 h and then washed three times in 0.9% NaC1 solution. Crude antigen was prepared by resuspending the cell-pellet in phosphate buffered saline (PBS; 10 m M phosphate, 0.15 M NaC1, pH 7.2) to 1 m g / m l followed by freezing. To prepare antigen for the monoclonal blocking ELISA, the cell-pellet was resuspended in 18 ml water and sonicated on ice for 45 sec. (100 W, 20 KC). Insoluble material was subsequently removed by centrifugation at 10 000 g for 15 min.
Rabbit antiserum. Polyclonal antibodies to M. hyopneumoniae were produced by immunization of rabbits with antigen harvested from rabbit broth cultures as described previously (Friis, 1977; Friis and Jensen, 1984). Serum from several bleedings was pooled and fractionated by a m m o n i u m sulphate precipitation (Harboe and Ingild, 1983 ), and the globulin fraction was used for coating the ELISA plates (see below).
Experimental infection of pigs. Reference sera to M. hyopneumoniae were prepared in 6 M. hyopneumoniae free, 12 week-old SPF pigs. Three of the pigs were infected once by aerosol inhalation for 7 min with 5 × 108 color changing units (ccu) of three pooled field strains of M. hyopneumoniae which had been filtered through a 0.45/~m filter and cloned once from solid medium. The other three pigs were contact infected by their penmates. All pigs were bled on the day of infection and then at weekly intervals for 3 months. To investigate cross reactions between M. hyopneumoniae and M. flocculore three caesarian section-derived, colostrum-deprived, 3 week-old piglets kept in isolation were infected (aerosol a n d / o r nasal instillation on days 0, 6,
MONOCLONAL BLOCKING ELISA TO MYCOPLASMA IIYOPNEU3,1ONIAE
37
35 and 41 ) with 107 ccu of a pool of two filter-cloned field strains of M.
flocculare. Production of monoclonal antibodies. Female BALB/c mice (8-12 weeks) were immunized subcutaneously three times three weeks apart with 200/tl of an equal mixture of M. hyopneumoniae crude antigen and Freund's incomplete adjuvant. The mice were boosted intravenously with 200/tl crude antigen three days before fusion. Spleen cells from immunized mice were fused with myeloma P3-X63-Ag8.653 cells (Kearny et al., 1979) using 50% (w/v) polyethylene glycol 4000 as described by Galfre and Milstein ( 1981 ), except that human endothelial culture supernatant (Costar, Badhoevedorp, Netherlands) was used instead of feeder cells (Astaldi et al., 1980). Growing hybridomas were screened by a competitive ELISA and cloned twice by limiting dilution. Briefly, competitive ELISA was performed essentially as described for blocking ELISA (see below), except that crude antigen was used for plate coating and mAb was demonstrated by incubation with horseradish peroxidase ( H P R ) conjugated rabbit anti-mouse immunoglobulin (Dako, Glostrup, Denmark). The binding of immunoglobulin from hybridoma culture supernatant (HCS) to antigen was inhibited by introducing a pool of sera from the pigs experimentally infected with M. hyopneumoniae as an intermediate layer. HCSs giving an absorbance, Apre, of more than 1.0 absorbance unit with pre-infection sera as the intermediate layer and less than 50% of Aore when the antibody-positive sera was used, were considered positive. Isotype and light chain of the mAb was determined in HCS by immunoblot assay as described by McDougal et al. (1983) using rabbit anti-isotype antisera and rabbit anti-light-chain antisera (Miles Scientific, Naperville, Illinois). The mAb was purified from HCS by affinity chromatography, using protein-A immobilized on agarose (Kem-En-Tec, Copenhagen, Denmark), and biotinylated according to the procedure described by Guesdon et al. ( 1979 ).
Immunoblotting. Crude antigen was submitted to electrophoresis according to standard procedures (Laemmli, 1970). The separated molecules were transferred to a 0.45/~m nitrocellulose membrane (Whatman, Maidstone, England) using a semidry electroblotter (Kem-En-Tec, Copenhagen, Denmark) as described by Kyhse-Andersen (1984). The transferred molecules were visualized either by staining with collodial gold (Moremans et al., 1985 ) or by immunological development essentially as described by Bjerrum et al. (1987). The molecular mass was estimated by comparison with known marker proteins: Ovalbumin (43.0 kDa), BSA (66.3 kDa), polymerase fl (150.6 kDa) and fl' ( 155.2 kDa) and myosin (approx. 200 kDa ) (Nene and Glass, 1984 ).
Dot blot assay. The species specificity of the mAb was investigated by a dot
38
N.C. F E L D ET AL.
blot assay using 20 field strains of M. hyopneumoniae, nine field strains plus one type strain (Ms42) of M. flocculare, seven field strains of M. hyorhinis, two field strains ofM. hyosynoviae, and one field strain ofM. dispar. Briefly, 105, 104 and 103 ccu of each strain was applied in a volume of 2 #1 to a nitrocellulose membrane and, after blocking with PBS containing 2% Tween 20 (PBS-T), the membrane was incubated for 1 h with a 1 : 10 dilution of HCS. Bound mAb was demonstrated as described for immunoblotting.
Blocking ELISA. The blocking ELISA was performed as follows: 96 well microtiter plates (Nunc, Roskilde, Denmark) were coated with 100/tl per well (all subsequent volumes are 100/~1 per well ) of rabbit immunoglobulin to M. hyopneumoniae diluted 1 : 1000 in 0.1 M carbonate buffer, pH 9.6. After incubation overnight at 4 ° C the plates were tapped dry and blocked for 1 h with PBS containing 4% (w/v)skim milk (PBS-M). The plates were washed in four changes of PBS-T followed by incubation for 1 h at room temperature with M. hyopneumoniae antigen diluted 1 : 250 in PBS-T. After another wash, serum samples diluted 1 in 10 in PBS-M were added in duplicate and the plates incubated for 2 h. PBS-M alone was applied to one row and both positive and negative control serum was added to four wells each. Without emptying, biotinylated mAb 17 diluted 1 : 2000 in PBS-M was added, and the plates were incubated for 15 min at room temperature. The plates were washed and incubated for 1 h with HRP-conjugated avidin (Dako, Glostrup, Denmark) diluted 1 : 8000 in PBS-T. After a final wash, enzyme substrate (8 mg 1,2orthophenyldiamine dihydrocloride, 12 ml 0.1 M citrate, pH 5 and 5 #1 H202 ) was added, and after 20 min colour development was stopped with 0.5 M H 2 S O 4. The spectrophotometric absorption was read at 490 nm using 650 nm as reference. The absorption mean of the row without pig serum was used for calculation of the percent inhibition by individual sera. The dilution and incubation time for biotinylated mAb 17 and pig serum were adjusted to give 0-20% inhibition with negative sera and 95-98% inhibition with sera from the pigs experimentally infected with M. hyopneumoniae. Preliminary 600 negative serum samples were investigated for confirming the adjustment. A test result was considered positive for inhibition >/50%.
IHA method. Fixed and tanned red blood cells (RBC) were sensitized with an extract of soluble antigens essentially as described by Holmgren (1974) with the modification that the RBC were fixed in pyruvic aldehyde (Rust et al., 1972 ). Complete agglutination of the RBC in serum dilution 1 : 80 or above was considered positive.
Field sera. Pig sera from SPF herds and SPF herds infected with M. hyopneumoniae were examined by blocking ELISA and by IHA. Group A consisted of 1000 sera obtained from 17 SPF herds without clinical signs of M. hyopneu-
MONOCLONAL BLOCKING ELISA TO MYCOPL,ISMA ltYOPNEUMONIAE
39
moniae infection but which occasionally had had a few reactors by IHA. Group B consisted of 498 serum samples from 46 SPF herds probably free of M. hyopneumoniae infection. Group C consisted of 462 samples from 23 SPF herds infected with M. hyopneumoniae and from which M. hyopneumoniae has been isolated. RESULTS
Monoclonal antibodies. A large number of hybridomas were identified but only supernatant from a single clone appeared positive in the competitive ELISA. This mAb, belonging to the IgG 1 t¢ isotype, was designated mAb 17. The molecular specificity of mAb 17 was demonstrated by immunoblotting (Fig. 1 ). When crude M.
A 1 2 3 4 5
B 1 2 3 4 5
C 1234
D 5
1
190 -122 - 94~ 74-64-49 4 4 --c 37--
Fig. I. Immunoblot analysis ofM. hyopneumoniae antigens using monoclonal antibody and sera collected sequentially from pigs infected with M. hyopneumoniae and M. flocculare. (A) Serum ( 1 : 100) from a pig experimentally infected with M. hyopneumoniae. Preinfection serum (A1) and serum obtained 2 weeks post infection (wpi) (A2), 4 wpi (A3), 6 wpi (A4) and 8 wpi (A5). (B) Serum ( 1 : 100) from a pig in contact with the pig in A above. Preinfection serum (B1) and serum obtained 2 wpi (B2), 4 wpi (B3), 6 wpi (B4) and 8 wpi (B5). (C) Serum ( 1 : 100) from a pig infected with M. flocculare. Preinfection serum (C 1) and serum obtained 2 wpi (C2), 4 wpi (C3), 6 wpi (C4) and 13 wpi (C5). (D) Hybridoma culture supernatant, mAb 17 ( 1 : 100), (D1).
40
N.C. F E L D E T A L .
hyopneumoniae antigen was used, mAb 17 recognized a molecule of approx. 74 kDa (Fig. 1, lane DI ). Four weeks after experimental infection with M.
hyopneumoniae, pig serum antibodies to molecules of approx. 122, 94, 74, 49 and 44 kDa were identified on immunoblots (Fig. 1, lane A3). After 8 weeks additional bands at approx. 190, 98, 93, 85, 80, 64, 52, 42 and 37 kDa were seen (Fig. 1, lane A5 ). The same pattern, although delayed by two weeks, was observed with serum from a pig kept in contact with the experimentally infected ones (Fig. 1), lanes B1-B5). Preimmune sera from the animals had antibodies to the 49 kDa molecule (Fig. l, lane A1 ) or both the 74 and 49 kDa molecules (Fig. 1, lane B1 ). These two patterns were also found in the other four pigs included in this experiment (data not shown). Eight weeks after infection with M. flocculare, antibodies to the 49 and 44 kDa molecules of M. hyopneumoniae were detected on immunoblots (Fig. 1, lane C4) and after 13 weeks antibodies to the 74 kDa molecule were also identified (Fig. 1, lane C5 ). Preinfection serum from this animal was negative by immunoblotting (Fig. 1, lane C 1 ). One of the two remaining caesarian section derived pigs confirmed this while preinfection serum from the last pig had antibodies to the 49 kDa molecules (data not shown).
Strain specificity ofmAb 17. The reactivity of mAb17 with other species of mycoplasma was investigated by dot blot assay, mAb 17 was shown to react with 103 ccu for all of the 20 field strains ofM. hyopneumoniae investigated. No reactions of mAb 17 were seen with the 105 ccu from any strain of M. flocculare, M. hyorhinis, M. hyosynoviae and M. dispar.
ELISA adjustment. In a preliminary study the mean inhibition percentage of 600 negative serum samples investigated over several days was 13.7 with a standard deviation of 10.3%. For the same investigation the inhibition of an internal positive standard was 70.7_+ 2.7%. The analytical sensitivity of the ELISA is mainly determined by the concentration and incubation time for the mAb but even a 10 min increase of incubation time only reduced the inhibition of the internal standard by 2.7%.
Experimentally infected pigs. All animals experimentally infected with M. hyopneumoniae showed a positive antibody response but at different intervals after infection. By ELISA the experimentally infected pigs showed a positive antibody response after two weeks. The titers peaked 8-9 weeks post infection and showed a slight decrease toward the end of the observation period. Contact infected pigs showed positive antibody response after 4-5 weeks followed by a rapid increase in titer so that the peak value and decline were mostly consistent with the values for experimentally infected pigs. A detectable antibody response was observed by IHA in the experimental pigs 2-5 weeks
M O N O C L O N A L B L O C K I N G ELISA T O
MYCOPLASMA HYOPNE[~,IONIAE
41
100"
.E
80"
_tJ
60"
//" /
/ /i II =-
20
g
/--'
t
"~
.,SJ
'"
0
1'o
o
1'2
1'4
weeks pest infection Fig. 2. Inhibition by sequential serum samples, diluted 1: 11, from pigs experimentally infected ( ) and contact infected (---) with M. hyopneumoniae.Day zero is the day of infection. A test result was considered positive for inhibition >/50%. after infection. For the contact infected pigs a positive antibody response was detected after 4-6 weeks. The m a x i m u m titer 1:2560 for experimentally infected pigs was identical in both assays. By IHA, for one pig the m a x i m u m titer was 80, while another showed the first detectable antibody response six weeks post infection. Serum from these pigs did not differ significantly by blocking ELISA from the other experimental pigs (Fig. 2 ). Three M. flocculare infected pigs having a homologous IHA titer i> 160 after 11 weeks showed no inhibition in the blocking ELISA during the investigation period.
Field samples. In group A serum samples, all were found to be negative in the blocking ELISA. One of the 17 herds had several reactors by IHA with titers >1640. M. flocculare was isolated from this herd. In group B one herd was found to be positive by both assays with eight reactors in ELISA and five reactors in IHA. This result was confirmed by isolating M. hyopneumoniae. The remaining herds were negative by ELISA but by IHA a single reactor was found in each of six herds. In the following three months no clinical sign of infection was noticed in these herds. In group C (Table 1 ) ELISA and IHA reactors were found in all except two herds which were negative by IHA. All samples positive by IHA were also positive by ELISA. In this group of chronically infected herds a total of 73 per cent of the samples tested positive by blocking ELISA compared with only 30 per cent by IHA. The ELISA inhibition m e a n for serum samples with IHA titer 80, 160 and >1320 was 84 + 12%, 92 _+8% and 94 ___3%, respectively.
42
N.C. FELD ET AL.
TABLE 1 Comparison o f blocking ELISA and IHA using sera from M. hyopneumoniae infected herds. Herd No.
No. tested ~
ELISA pos. 2
Mean inhibition ELISA pos. (S.D.)3
no. (%) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 TOTAL
18 20* 20* 20 21" 20 20* 30* 20* 20 20 19" 19" 21 20* 18"* 20 20* 19 19" 19 19" 20 462
12 18 11 16 17 11 15 23 19 14 15 17 9 14 19 10 9 16 16 16 15 14 9 335
(67) (90) (55) (80) (81) (55) (75) (77) (95) (70) (75) (89) (47) (67) (95) (56) (45) (80) (84) (84) (79) (74) (45) (73)
78.8 73.7 71.5 88.3 74.7 72.5 77.8 83.7 83.5 71.4 75.5 91.2 70.0 83.8 93.8 66.9 77.9 82.8 83.4 77.5 74.1 76.7 70.9
(17.5) (12.9) (13.3) (9.3) (11.4) (15.2) (11.2) (8.9) (14.0) (13.7) (13.4) (4.6) (12.8) (14.1) (5.8) (12.0) (16.7) (17.1) (12.1) (13.0) (16.9) (14.1) (15.2)
IHA
pos. 4
Distribution o f 1HA titer 5
no. (%)
80
160
>/320
7 (39) 9 (47) 3 (16) 11 (55) 6(30) 3(15) 5 (26) 12 (41) 13 (68) 2 (10) 3(15) 3 (17) (0) 6 (29) 11 (58) (0) 7 (35) 11 (58) 5 (26) 3 (17) 1 (5) 7 (39) 6 (30) 132 (30)
4 5 2 2 5 1 4 10 3 2 1 2
2 3 1 1 1 1 2 1 1
3 2
2 3
5 3 4 3 4 4
3 1 2 1
1 l 8 I 1 2 8 1 1 6 2 4 1 1 1
*One sample not testable by IHA due to spontaneous hemagglutination. ~The n u m b e r o f serum samples tested in each herd. 2The n u m b e r o f reactors found by ELISA and (percent). 3The ELISA mean inhibition of positive samples for each herd and the standard deviation in parenthesis. 4The n u m b e r o f reactors found by IHA and (percent). SThe IHA reactors distributed by titer.
DISCUSSION
The aim of the present project was to develop a specific and sensitive serological ELISA based on monoclonal antibody competing with natural antibodies to M. hyopneumoniae in porcine serum, mAb 17 appeared to be the first monoclonal antibody with this property to be described for M. hyopneumomiae immunoassays. It was demonstrated that mAb 17 reacted with a molecule banding at approx. 74 kDa after SDS-PAGE and immunoblotting. Mori et al. ( 1988 ) demonstrated antibodies to the 74 kDa molecule in por-
MONOCLONAL BLOCKING ELISA TO MYCOPLASMA HYOPNEL~VIONIAE
43
c~ne hy-perimmune serum to M. hyopneumoniae, M. flocculare and M. hyor~hinis. Others found molecules of approx, this mass in all three mycoplasmas (B/51ske et al., 1987). This reactivity appears to involve epitopes other than the one recognized by mAb 17 because only porcine antiserum to M. hyopneumoniae was able to inhibit the binding of mAb 17 in competitive ELISA. The experimental pigs were from a certified herd and hardly sensitized to M. hyopneumoniae before the experimental exposure. The positive reaction in immunoblot of the preinoculation sera could represent cross-reactions with other microorganisms. Recently, it was reported that the gene coding for a 74 kDa protein shared sequence similarity with DnaK ofEscherichia coli (Birkel a n d et al., 1990). Sequences from this protein, one of the family of highly conserved 70 kDa heat shock proteins, seems to be expressed by a variety of microorganisms (Topp and Faulds, 1990), and this could give rise to the reactions observed by immunoblotting. Most of the cae arian section derived, colostrum deprived pigs were probably not colonized by a large number of different microorganisms and therefore were negative in immunoblotting. Nicolet et al. (1980) found that the use of ultrasonically disintegrated M. hyopneumoniae antigen in an ELISA caused non-specific binding of porcine immunoglobulins and Mori et al. (1987) observed non-specific binding of mAbs (especially IgG isotypes) to porcine immunoglobulins. Our prelimi.nary results confirm these findings but by the use of rabbit immunoglobulin as the catching antibody in the ELISA, non-specific binding of mAb 17 (IgG1 to) was eliminated. Sera from experimentally infected pigs and from herds with known M. hyopneumoniae status were compared by IHA and ELISA for evaluation of the blocking ELISA. Using the calculated inhibition percentage for experimental animals, a clear distinction between inoculated and contact infected animals was observed (Fig. 2 ). Using the ELISA, experimental infected pigs were found to have seroconverted between the first and the second week. This time of seroconversion is among the earliest reported in literature for M. hyopneumoniae immunoassays (Lloyd et al., 1987). In contrast to Armstrong et al. (1983) we found no difference in titer between experimental infected and contact infected pigs. A gradual decrease in titer observed in experimental pigs after week 9 (results not shown ) was hardly visible from the inhibition curve (Fig. 2 ). Due to the difficulty of isolating M. hyopneumoniae, the assays were compared using test-samples from SPF-herds infected with M. hyopneumoniae. This comparison showed a significantly increased number of reactors for the ELISA (Table 1 ). Neither the weak, nor the slow IHA responding pig were significantly different from the other experimental pigs when examined by ELISA. The negative IHA result observed in two of the herds in group C may be due to the time of seroconversion detected by this assay. In chronically infected herds, piglets are protected by maternal antibodies for the first cou-
44
N.C. FELD ET AL.
ple of months and antibodies due to infection are first detected at 90-150 days of age (Mori et ah, 1987). The time of seroconversion is dependent on farm management, infection pressure and the assay used. Test-samples in this investigation were from 3-5 month old pigs. The specificity of M. hyopneumoniae immunoassays is often evaluated with sera from pigs experimentally infected with other mycoplasmas. In agreement with Armstrong et al. ( 1987 ) we found that pigs experimentally infected with M. flocculare were slow to seroconvert and when they did, titers were low and antibodies to M. hyopneumoniae were not detectable. In contrast, we found by IHA some naturally M. flocculare-infected herds (M. flocculare isolated) with high-titered cross-reacting antisera to M. hyopneumoniae. The herds with few reactors by IHA (group A and B) were intensively surveyed at the slaughter line and by clinical inspection in the herds but no history ofM. hyopneumoniae infection could be verified. As quoted by others (Armstrong et al., 1983) IHA is not an ideal assay for surveillance ofM. hyopneumoniae infection because ofunspecificity and difficulty in the reproducibility of alike antigen coated RBC. The described blocking ELISA may prove to be an advantage in the serodiagnostic and surveillance of M. hyopneumoniae infections in pigs. ACKNOWLEDGEMENTS
We thank DVM Kristen Barfod and DVM Vibeke Sorensen, The Federation of Danish Pig Producers and Slaughterhouses (Vet. Dept.), Roskilde, Denmark, for kindly supplying us with information and sera from SPF pig herds. This work was supported in part by the Danish Agricultural and Veterinary Research Council.
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B61ske, G., Strandberg, M., Bergstr6m, K. Johansson, K., 1987. Species-specific antigens of Mycoplasma hyopneumoniae and cross-reactions with other porcine mycoplasmas. Curr~ Microbiol. 15: 233-239. Boulanger, P. and L'Ecuyer, C., 1968. Enzootic pneumoniae of pigs: Complement-fixation tesi for the detection of mycoplasma antibodies in the serum of immunized rabbits and infected' swine. Can. J. Comp. Med., 32: 547-554. Bruggmann, S., and Keller, H., 1977. Quantitative detection of antibodies to Mycoplasma suipneumoniae in pigs' sera by an enzyme-linked immunosorbent assay. Vet. Rec., 101: 109111. Friis, N.F., 1977. Mycoplasma suipneumoniae and Mycoplasmaflocculare in the growth precip, itation test. Acta Vet. Scand., 18:168-175. Friis, N.F., and Jensen, P.T., 1984. Serological comparison of type strains of porcine, bovine; and ovine mycoplasmas with atypical colony morphology. Acta Vet. Scand., 25: 29-35. Galfrb, G., and Milstein, C., 1981. Preparation of monoclonal antibodies: Strategies and pro-' cedures. Methods. Enzymol., 73: 1-46. Goodwin, R.F.W., Hodgson, R.G., Whittlestone, P. and Woodhams, R.L., 1969. Immunity in experimentally induced enzootic pneumoniae of pigs. J. Hyg. Camb., 67:193-207. Guesdon, J.-L., Ternynck, T. and Avrameas, S., 1979. The use of avidin-biotin interaction in, immunoenzymatic techniques. J. Histochem. Cytochem., 27:1131-1139. Harboe, N.M.G. and Ingild, A., 1983. Immunization, isolation of immunoglobulins and antibody titre determination. Scand. J. lmmunol., 17:345-351. Holmgren, N., 1974. An indirect hemagglutination test for detection of antibodies against Mycoplasma hyopneumoniae using formalized tanned swine erythrocytes. Res. Vet. Sci., 16: 341-346. Hovind-Hougen, K. and Friis, N.F., 1991. Morphological and ultrastructural studies of Mycoplasrnaflocculare and Mycoplasma hyopneumoniae in vitro. Res. Vet. Sci., in press. Kearny, J.F., Radbruch, A., Liesegang, B. and Rajewsky, K., 1979. A new mouse myeloma cell line that lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J. Immunol., 123:1548-1550. Kyhse-Andersen, J. 1984. Electroblotting of multiple gels: A simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J. Biophys. Biochem. Methods, 10: 203-209. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly c~fthe head of bacteriophage T4. Nature (London), 227: 680-685. Lloyd, L.C., Cottew, G.S. and Anderson, D.A., 1987. Early serological responses to Mycoplasma hyopneumoniae infection. Israel J. Med. Sci., 23: 647-649. McDougal, J.C., Browning, S.W., Kennedy, S. and Moore, D.D., 1983. lmmunodot assay fordeterming the isotype and light chain of murine monoclonal antibodies in unconcentrated hybridoma culture supernatants. J. Immunol. Methods, 63:281-290. Moremans, M., Daneels, G. and De Mey, J., 1985. Sensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes. Anal. Biochem., 145:315-321. Mori, Y., Hamaoka, T. and Sato, S., 1987. Use of monoclonal antibody in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Mycoplasma hyopneumoniae. Israel J. Med. Sci., 23: 657-662. Mori, Y., Hamaoka, T., Sato, S. and Takeuch, S., 1988. Immunoblotting analysis of antibody response in swine experimentally inoculated with Mycoplasma hyopneumoniae. Vet. Immunol. Immunopathol., 19: 239-250. Nene, V. and Glass, R.E., 1984. Genetic studies on the fl subunit ofEscherichia coli RNA polymerase. Mol. Gen. Genet., 194:166-172.
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Nicolet, J., Paroz, P. and Bruggmann, S., 1980. Tween 20 soluble proteins ofMycoplasma hyopneumoniae as antigen for an enzyme-linked immunosorbent assay. Res. Vet. Sci., 29: 305309. Rust, J.H., Berman, S., Habig, W.H., Marshall, J.D. and Cavanaugh, D.C., 1972. Stable reagent for the detection of antibody to the specific fraction 1 antigen of Yersinia pestis. Appl. Microbiol., 93: 721-724. Topp, C., and Faulds, D.H., 1990. Cloning and nucleotide sequence of the gene for the 74.5 kDa antigen, and HspT0 homologue in Mycoplasma hyopneumoniae. Abstr. 8th Int. Congr. IOM, Istanbul, Turkey, IOM lett., 1: 270-271.
