Immunology Letters, 23 (1989/1990) 217- 222 Elsevier IMLET 01337
Immunological specificity of monoclonal antibodies to Chlamydia psittaci ovine abortion strain H. Puy, V. Fuentes, E E b and J. O r f i l a Laboratoire de Bact~riologie-lmmunologie G~n~rale, Centre Hospitalo-Universitaire
d'Amiens, Amiens, France
(Received 16 Oclober 1989; accepted 30 October 1989)
1. Summary Fifty-one monoclonal antibodies were prepared by two different techniques against Chlamydia psittaci strain A22 isolated from an ovine abortion. These antibodies were tested for reactivity by the indirect immunofluorescent antibody technique with eleven reference Chlamydia strains (nine C. psittaci, one Chlamydia trachomatis and one Chlamydia
pneumoniae). Four classes of specificity were recognized for monoclonal antibodies: genus, species, subspecies and type specificity. The type-specific monoclonal antibodies were non-reactive with ovine arthritis isolates. Twenty monoclonal antibodies were specific for two mammalian strains: ovine abortion A22 and K mouse. Some monoclonal antibodies were reactive with C. pneumoniae strains and non-reactive with C. trachomatis strains. All these monoclonal antibodies were very useful for improving the diagnosis of chlamydial infection, the antigenic analysis and the serotyping of C. psittaci.
against C psittaci, essentially avian strains [1-5]. In this study, monocional antibodies were prepared against a mammalian C. psittaci strain (A22) isolated from an ovine abortion, and the reactivities of the antibodies to several strains of C psittacL C trachomatis and C. pneumoniae were analyzed. The recent discovery in our laboratory of crossimmunity between this ovine abortion strain and avian strain [6] justified the utilization of the A22 strain for this study.
3. Materials and Methods
3.1. Chlamydial strains Strain A22, isolated from an ovine abortion (J. Treharne, Ophthalmology Institute, London, U.K.) and grown in L 929 cells, was employed to immunize mice. The eleven strains of Chlamydia used for the screening of monoclonal antibodies and grown in the yolk sacs of chicken eggs are listed with their host in Table 1.
2. Introduction
3.2. Mice and immunization
I f the genus C. trachomatis is classified into 15 immunotypes, C. psittaci is still not classified completely into distinct immunotypes. Recently, several authors have produced monoclonal antibodies
BALB/c female mice were used, as these mice are not fatally susceptible to A22 strain and are high humoral immune responders. Mice received by intraperitoneai injection 0.5 ml of infectious suspensions titrated at 7×106 inclusion-forming units by the method of Fuentes [7]. A booster immunization was performed after lbur weeks, and three days later the spleen was harvested for fusion.
Key word.v Chlamydia psittaci; Monoclonal antibody Correspondence to." H. Puy, Laboratoire de Bacteriologielmmunologie g6n6rale, Centre Hospitalo-Universitaire d'Amiens, Place Victor Pauchet 80030 Amiens, France.
0165-2478 / 90 / $ 3.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
217
3.3. Cell fltsion
3.4. Characterization and screening q f m o n o c l o n a l antibodies
S P 2 0 a n d X 63-Ag 8653 m y e l o m a cells were fused with i m m u n e splenocytes using P E G 4000 with the standard procedure [8]. Stable hybrids were selected in H A Z (hypoxanthine a n d azaserine) m e d i u m for S P 2 0 myeloma cells and in HAT (hypoxanthine, a m i n o p t e r i n e a n d t h y m i d i n e ) m e d i u m for X 63-Ag 8653 myeloma cells. H y b r i d o m a cells p r o d u c i n g antibodies were cloned.
The m i c r o - i m m u n o f l u o r e s c e n c e techniquc of Wang a n d Grayston [9] was used to detect and determine the specificity o f m o n o c l o n a l a n t i b o d i e s on a panel of eleven classified referenced C h l a m y d i a strains. The j u d g e m e n t of a positive reaction was based on definite fluorescence at the site of the specific particular antigen. The isotypicity of m o n o c l o n a l a n t i b o d i e s was d e t e r m i n e d by a d o u b l e i m m u n o d i f f u s i o n against a n t i - m o u s e -,/1, -;2a, ?2b, 3,3 and ,u antisera (provided by Cappel).
IABLE I Isolates of Chlamydia used in this stud}'. Chlamydial isolate
Species
Hosl
Disease
Origin
I_BI
C. trachomatis
Human
Cervicitis
1)r. Hanna San Francisco (USA}
IOI.207
C. pneumonia (IWAR strain)
Human
Conjunctivitis
Prof. J. D. lreharne London (GB)
AIO
C. psittaci
Guinea pig
Conjunctivitis
Prof. J. I). Treharne
t,ondon (GB) A22
C. pstttact
Sheep
Abortion
Prof. J. D. Treharne London ((iB)
457 cat
C. pslttact
Cat
Conjunctivitis
F'rof. J. 1). Treharne London {GB)
I'P 145
C. p,stttact
Cat
Pneumonia
Prof. J. Schachter San Francisco (USA}
1.W679
C. pstttact
Sheep
Polvarthrifis
F'rof. J. Storz Fort Collins (LISA)
MPI
C. psttlact
Mouse
Meningopneumonia
Prof. A. lamura Kyoto (Japan)
TI3
C. pstttact
Parakeet
Psittacosis
Dr. F. Edlinger Paris (France)
Loth
C. pstll~lcI
Pigeon
Ornithosis
Prof. Dekking .Amsterdam (The Netherlands)
K mouse
C. psittaci
Mouse
lnapparent respiratory infection
Prof..I. Storz Fort Collins (USA)
218
4.
Results
The Ag
and
results
8653
Discussion
are
immunization
summarized
technique
arid
in Table in
Table
2 for
3
for
the
the
X63-
SP20
The
specificity
(35
or
isotypicity to
that
of these found
51 monoclonal
in
other
antibodies
studies
using
of
similar
seem
Of
to
were
subspeet al.
from
14 were
with
a limited
Andersen
antibodies
these,
agree
a high
with
antibodies
Recently,
16 monoclonal
results
we obtained
antibodies
monoclonal
psittaci.
C.
[1], but
monoclonal
type-specific).
prepared
is close
TABLE
of
cies
technique.
procedures
proportion
[10]
4 isolates
type-specific;
their
ours.
2 Isotypicity
Antibodies
Heavy chain
and
specificity
C. trachomatis
of 37 monoclonal
antibodies
produced
with
X63 - Ag 8659 myeloma
cells.
C. psittaci Avian
C. pneumoniae
strains
Mammalian
strains
IOL
207
LBI Loth IA6
"),2a
.
IA8
y2a
+ +
.
IB4
y2a
.
IB7
3,2a
.
IB9
yl
.
IBI0
ND
-
1(_'4
")'2b
-
ID5
~2a
.
ID8
",:2a
.
IDI2
y2a
.
IE6
y2a
.
IE8
yl
IEII
ND
* + +
IEI2
ND
.
IF3
~1
IF7
y2a
IF8
y2a
IFI2
y2a
-
165
ND
++
.
.
llC2
ND
K mouse
~- + 4-
--
* +
+ +
+ +
+ +
+ * *
* 4-
+
+
. .
.
+++
.
.
.
.
.
.
. .
.
.
+ + +
~- + 4.
.
.
. 4-++ .
.
+++
++
.
4- 4-
+ +
+ + +
+ +
++
+ 4-
.
-,- +
.
IIEll
-~2b
.
IIFII
"t2a
-
IIG3
3'1
.
IIGII
y2a
++
+++
++
++
++
-~ +
+++
IIICI2
y2a
-
+ + +
+ + +
+ +
+ 4.
+ +
* ~-
IIIDI0
"t2a
.
IIIGI2
"r2a
.
IVCII
3,2a
.
IVDI2
3,3
IVF9
y2a
.
.
.
.
. .
+ 4- 4-, H i g h
.
. .
.
.
.
.
.
.
.
.
.
.
.
.
. .
titer by the microimmunofluorescence
+
-
+
* *
-
+ + +
--
+
-
4. + +
-
+ 4
-
++
. . . .
4-
+ + +
+ +
+++
+ ++
+.-4-
+ +
+ ~
_
4-
+ 4- 4-
-
* +
+ *
-
+ ~--
4 4- +
+ +
+ .
~- + q-
+
~- + 4.
+
, *
-
+ 4-
.
+
+
+ + +
+ 4 +
+ + +
~-
~-4-+
+ ++
+4
-~ 4-
.
+ + +
+ +
. . . technique;
--
+ *
. .
++
+ +
-
.
. .
+ ÷+
+4-+
.
. .
+
+ ++
4- -
.
. .
.
.
.
. .
.
-
.
. .
.
.
.
.
. .
.
-
.
.
.
.
+ + +
+
.
.
.
+ +
3,2a
.
-
+
+ +
+ +
.
+
+ +
.
y2a
.
-
.
lIE6
+ +
+
-
+ +
+
+ +
~-++
IIEI0
.
.
4-
. .
.
-
.
+ +
. .
~-
. .
+ +
. .
+
4-+
.
.
+ .
+++
. .
.
-
+ + +
. . . .
+
~*
+ +~
+
+ + +
-
+ + +
+++
+
+
-
4-++
.
--
~ ~
A22
+
-
+ + ~
.
.
.
. .
+ + +
++4.
.
+++
.
.
+++
.
+
.
.
4++
+
.
4-:,-*
+
+ 4.
+
+
+-~-
.
.
.
+4.
+
+
. .
+ ++
. . . .
.
.
.
+++
.
+
. .
.
+ ++
*
.
. .
++
.
.
.
*÷
.
.
.
**
++*
.
4- +
+ + -
+ +
y2a
LW679
-
.
lIB8
MPI
.
.
.
.
AI0
+ +
.
.
~3 y2a
.
+ -~
.
",/2b y2a
Cat 457
.
+ +
.
IG6 IH8
FPI45
.
+ +
IH4 IIA3
TI3
+ 4-, m c d i u m
titer;
+,
+ +
+ +
4- + +
+ ~ -
-
+ 4.
4 + +
-
+ * +
+ + +
-
4- + +
.
~- +
-
4- + +
+ ~ +
-
lov,' t i t e r ;
-,
+
negative.
219
TABLI.
3
lsolypicity and specificity of 14 m o n o c l o n a l antibodies produced v.'ith SP20 myeloma cells. Antibodies
Heavy
C.
chain
matis
tracho-
('. ])?lClgmOtli~lC
('. p s i t t a c i .
Avian strains
.
.
-
"tl
4
NI) ~ll
.
.
.
TI3
I.P145
(7at 457
A10
.
MPI
.
I.W679
K mouse
~
+
-~-
a
r~
* -
4 * t
t
~
.-
--
T
1
*
t
, + t
t-
..
~-
1
4-
t-
• + ÷.
~
a.
r
4-
t
4-
t
4-{"
I + ~-
•
• +
t
a. +
~--~ a-
-:-
t
4
i
+
t
4-
~ 4-
t
.~
~-
~
k-
4-
t
4-
~
t" 4
f
a
.
tt
f * ~ 4
k+
~-
t
4
4-
t-
a
~
~
~-
t
4-
.
,-
I
4-
4
t
,1
4-
~-
4
4
I
~
~-
-rl
We found good correlation with preceding work on serotyping of C. psittaci using polyclonal sera [11-14]. Twenty monoclonal antibodies had a crossspecificity between the ovine abortion strain A22 and another mammalian strain: K mouse• Eb [11], using polyclonal sera, had already described the high h o m o l o g y of antigenic structure between these two mammalian strains• On the other hand, we did not find any cross-specificity between two ovine strains which were pathogenically different (arthritis and abortion isolates). This is in accordance with De Long and Magee [15], who reported a monoclona[ antibody which was specific to an ovine abortion strain of C. psittaci and non-reactive with the ovine arthritis isolates. Finally, five monoclonal antibodies reacted to all C. psinaci strains and to the third species of Chlarnydia: C. pneumoniae (TWAR strain) but were non-reactive to C. trachomatis (LBI strain)• (7. pneumonia, the third species in the genus Chlarnydia on the basis of molecular biology [16, 17], was first classified as a C. psittaci strain because of the similarity in inclusion morphology [18]. We thought that this strain was also nearer to (7..psittaci than to (7.. trachomalts in antigenic structure. These results confirm the molecular biology and
t
-~
~ -
4
-t
4
t-
4
1 -
f-
t
~
1-
t
4
a-
-.
4"
l-
4-
t
4
"I-
T
+ 4-, High titer by the m i c r o i m n m n o f l u o r e s c e n c e technique; o, +, medium liter; + , l o w l i t c r ;
220
A22
44-
31 NI) 73 ~[1
.
.
•
"t2b
.
IO1. 2(.)7 .
Lolh *t2a "tl
.
M a m m a l i a n strains
1 B1
A5 AI7 AI9 A20 A21 A22 A23 B5 B6 BI2 BI3 BI6 B19 B22
.
•
4
, negative.
immunology studies; monoclonal antibodies appear to be a very good tool, capable of improving our knowledge of epidemiology and pathogenicity in
Chlamydia. References 111 Fuentes, V., l.efebvre, .I.I.., l e m a , F., Bissac, E. and Orfila, .I. (1985) l m m u n o l , l.ett. 10, 325. [21 Pakahashi, T., Takashima, 1. and Hashimolo, N. (1988) Microbioh l m m u n o l . 32, 251. [3] Seki, C., l a k a s h i m a , I., Arikav, a, J. and Hashirnoto, N (1988) .Ipn. J. Vet. Sci. 50, 383. [4] Fukushi, H., Nojiri, K. and Hirai, K. (1987).I. ( l i n . Microbiol. 25, 1978. 15] Toyofuku, H., l a k a s h i m a , 1., Arikawa, J. and Hashimoto, N. (1986) Microbiol. lmmunol. 30, 945. [6] Fuentes, V., Puy, H., Lefebvre, J. F. and Orfila, J. (1989) Microhios I.ett. 40, 55. [7] Fuentes, V., I-b, F. and Orfila, J. (1988) J. P,iol. Stand. 16, I. [8] Kohler, G. and Milstein, C. (1979) Nature 296, 495. [91 Wang, S. P. and Grays/on, .1. I. (1970) Am..I. O p h t h a h n o l . 70, 367. [10] Andersen, A. and Van Deusen, R. (1988) Infect. h n m u n . 56, 2075. [111 Eb, I..,Orfila,.I., Milon, A. and (ieral, M. I-.(1986) Ann. Inst. Pasteur Microbiol. 137 B, 77. [12] Perez-Martinez, J. A. and Storz, J. (1985) Infect. l m m u n . 50, 905.
[13] Schachter, .I., Banks, J., Sugg, N., Sung, M., Storz, J. and Meyer, K. E (1974) Infect. lmmun. 9, 92. [14] Schachter, J., Banks, J., Sugg, N., Sung, M., Storz, J. and Meyer, K. E (1975) Infect. lmmun. I1,904. [15] Delong, W. J. and Magee, W. E. (1986) Am. J. Vet. Res. 47, 1520.
[16] Cox, R. L., Kuo, C. C., Grayston, J. T. and Campbell, L. A. (1988) Int. J. Syst. Bacteriol. 38, 265. [17] Grayston, J. T., Kuo, C. C., Campbell, L. A. and Wang, S. P. (1989) Int. J. Syst. Bacteriol. 39, 88. [18] Grayston, J. T., Kuo, C. C., Wang, S. P. and Altman, J. (1986) New Engl. J. Med. 315, 161.
221
Immunological specificity of monoclonal antibodies to Chlamydia psittaci ovine abortion strain H. Puy, V. Fuentes, E E b and J. O r f i l a Laboratoire de Bact~riologie-lmmunologie G~n~rale, Centre Hospitalo-Universitaire
d'Amiens, Amiens, France
(Received 16 Oclober 1989; accepted 30 October 1989)
1. Summary Fifty-one monoclonal antibodies were prepared by two different techniques against Chlamydia psittaci strain A22 isolated from an ovine abortion. These antibodies were tested for reactivity by the indirect immunofluorescent antibody technique with eleven reference Chlamydia strains (nine C. psittaci, one Chlamydia trachomatis and one Chlamydia
pneumoniae). Four classes of specificity were recognized for monoclonal antibodies: genus, species, subspecies and type specificity. The type-specific monoclonal antibodies were non-reactive with ovine arthritis isolates. Twenty monoclonal antibodies were specific for two mammalian strains: ovine abortion A22 and K mouse. Some monoclonal antibodies were reactive with C. pneumoniae strains and non-reactive with C. trachomatis strains. All these monoclonal antibodies were very useful for improving the diagnosis of chlamydial infection, the antigenic analysis and the serotyping of C. psittaci.
against C psittaci, essentially avian strains [1-5]. In this study, monocional antibodies were prepared against a mammalian C. psittaci strain (A22) isolated from an ovine abortion, and the reactivities of the antibodies to several strains of C psittacL C trachomatis and C. pneumoniae were analyzed. The recent discovery in our laboratory of crossimmunity between this ovine abortion strain and avian strain [6] justified the utilization of the A22 strain for this study.
3. Materials and Methods
3.1. Chlamydial strains Strain A22, isolated from an ovine abortion (J. Treharne, Ophthalmology Institute, London, U.K.) and grown in L 929 cells, was employed to immunize mice. The eleven strains of Chlamydia used for the screening of monoclonal antibodies and grown in the yolk sacs of chicken eggs are listed with their host in Table 1.
2. Introduction
3.2. Mice and immunization
I f the genus C. trachomatis is classified into 15 immunotypes, C. psittaci is still not classified completely into distinct immunotypes. Recently, several authors have produced monoclonal antibodies
BALB/c female mice were used, as these mice are not fatally susceptible to A22 strain and are high humoral immune responders. Mice received by intraperitoneai injection 0.5 ml of infectious suspensions titrated at 7×106 inclusion-forming units by the method of Fuentes [7]. A booster immunization was performed after lbur weeks, and three days later the spleen was harvested for fusion.
Key word.v Chlamydia psittaci; Monoclonal antibody Correspondence to." H. Puy, Laboratoire de Bacteriologielmmunologie g6n6rale, Centre Hospitalo-Universitaire d'Amiens, Place Victor Pauchet 80030 Amiens, France.
0165-2478 / 90 / $ 3.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
217
3.3. Cell fltsion
3.4. Characterization and screening q f m o n o c l o n a l antibodies
S P 2 0 a n d X 63-Ag 8653 m y e l o m a cells were fused with i m m u n e splenocytes using P E G 4000 with the standard procedure [8]. Stable hybrids were selected in H A Z (hypoxanthine a n d azaserine) m e d i u m for S P 2 0 myeloma cells and in HAT (hypoxanthine, a m i n o p t e r i n e a n d t h y m i d i n e ) m e d i u m for X 63-Ag 8653 myeloma cells. H y b r i d o m a cells p r o d u c i n g antibodies were cloned.
The m i c r o - i m m u n o f l u o r e s c e n c e techniquc of Wang a n d Grayston [9] was used to detect and determine the specificity o f m o n o c l o n a l a n t i b o d i e s on a panel of eleven classified referenced C h l a m y d i a strains. The j u d g e m e n t of a positive reaction was based on definite fluorescence at the site of the specific particular antigen. The isotypicity of m o n o c l o n a l a n t i b o d i e s was d e t e r m i n e d by a d o u b l e i m m u n o d i f f u s i o n against a n t i - m o u s e -,/1, -;2a, ?2b, 3,3 and ,u antisera (provided by Cappel).
IABLE I Isolates of Chlamydia used in this stud}'. Chlamydial isolate
Species
Hosl
Disease
Origin
I_BI
C. trachomatis
Human
Cervicitis
1)r. Hanna San Francisco (USA}
IOI.207
C. pneumonia (IWAR strain)
Human
Conjunctivitis
Prof. J. D. lreharne London (GB)
AIO
C. psittaci
Guinea pig
Conjunctivitis
Prof. J. I). Treharne
t,ondon (GB) A22
C. pstttact
Sheep
Abortion
Prof. J. D. Treharne London ((iB)
457 cat
C. pslttact
Cat
Conjunctivitis
F'rof. J. 1). Treharne London {GB)
I'P 145
C. p,stttact
Cat
Pneumonia
Prof. J. Schachter San Francisco (USA}
1.W679
C. pstttact
Sheep
Polvarthrifis
F'rof. J. Storz Fort Collins (LISA)
MPI
C. psttlact
Mouse
Meningopneumonia
Prof. A. lamura Kyoto (Japan)
TI3
C. pstttact
Parakeet
Psittacosis
Dr. F. Edlinger Paris (France)
Loth
C. pstll~lcI
Pigeon
Ornithosis
Prof. Dekking .Amsterdam (The Netherlands)
K mouse
C. psittaci
Mouse
lnapparent respiratory infection
Prof..I. Storz Fort Collins (USA)
218
4.
Results
The Ag
and
results
8653
Discussion
are
immunization
summarized
technique
arid
in Table in
Table
2 for
3
for
the
the
X63-
SP20
The
specificity
(35
or
isotypicity to
that
of these found
51 monoclonal
in
other
antibodies
studies
using
of
similar
seem
Of
to
were
subspeet al.
from
14 were
with
a limited
Andersen
antibodies
these,
agree
a high
with
antibodies
Recently,
16 monoclonal
results
we obtained
antibodies
monoclonal
psittaci.
C.
[1], but
monoclonal
type-specific).
prepared
is close
TABLE
of
cies
technique.
procedures
proportion
[10]
4 isolates
type-specific;
their
ours.
2 Isotypicity
Antibodies
Heavy chain
and
specificity
C. trachomatis
of 37 monoclonal
antibodies
produced
with
X63 - Ag 8659 myeloma
cells.
C. psittaci Avian
C. pneumoniae
strains
Mammalian
strains
IOL
207
LBI Loth IA6
"),2a
.
IA8
y2a
+ +
.
IB4
y2a
.
IB7
3,2a
.
IB9
yl
.
IBI0
ND
-
1(_'4
")'2b
-
ID5
~2a
.
ID8
",:2a
.
IDI2
y2a
.
IE6
y2a
.
IE8
yl
IEII
ND
* + +
IEI2
ND
.
IF3
~1
IF7
y2a
IF8
y2a
IFI2
y2a
-
165
ND
++
.
.
llC2
ND
K mouse
~- + 4-
--
* +
+ +
+ +
+ +
+ * *
* 4-
+
+
. .
.
+++
.
.
.
.
.
.
. .
.
.
+ + +
~- + 4.
.
.
. 4-++ .
.
+++
++
.
4- 4-
+ +
+ + +
+ +
++
+ 4-
.
-,- +
.
IIEll
-~2b
.
IIFII
"t2a
-
IIG3
3'1
.
IIGII
y2a
++
+++
++
++
++
-~ +
+++
IIICI2
y2a
-
+ + +
+ + +
+ +
+ 4.
+ +
* ~-
IIIDI0
"t2a
.
IIIGI2
"r2a
.
IVCII
3,2a
.
IVDI2
3,3
IVF9
y2a
.
.
.
.
. .
+ 4- 4-, H i g h
.
. .
.
.
.
.
.
.
.
.
.
.
.
.
. .
titer by the microimmunofluorescence
+
-
+
* *
-
+ + +
--
+
-
4. + +
-
+ 4
-
++
. . . .
4-
+ + +
+ +
+++
+ ++
+.-4-
+ +
+ ~
_
4-
+ 4- 4-
-
* +
+ *
-
+ ~--
4 4- +
+ +
+ .
~- + q-
+
~- + 4.
+
, *
-
+ 4-
.
+
+
+ + +
+ 4 +
+ + +
~-
~-4-+
+ ++
+4
-~ 4-
.
+ + +
+ +
. . . technique;
--
+ *
. .
++
+ +
-
.
. .
+ ÷+
+4-+
.
. .
+
+ ++
4- -
.
. .
.
.
.
. .
.
-
.
. .
.
.
.
.
. .
.
-
.
.
.
.
+ + +
+
.
.
.
+ +
3,2a
.
-
+
+ +
+ +
.
+
+ +
.
y2a
.
-
.
lIE6
+ +
+
-
+ +
+
+ +
~-++
IIEI0
.
.
4-
. .
.
-
.
+ +
. .
~-
. .
+ +
. .
+
4-+
.
.
+ .
+++
. .
.
-
+ + +
. . . .
+
~*
+ +~
+
+ + +
-
+ + +
+++
+
+
-
4-++
.
--
~ ~
A22
+
-
+ + ~
.
.
.
. .
+ + +
++4.
.
+++
.
.
+++
.
+
.
.
4++
+
.
4-:,-*
+
+ 4.
+
+
+-~-
.
.
.
+4.
+
+
. .
+ ++
. . . .
.
.
.
+++
.
+
. .
.
+ ++
*
.
. .
++
.
.
.
*÷
.
.
.
**
++*
.
4- +
+ + -
+ +
y2a
LW679
-
.
lIB8
MPI
.
.
.
.
AI0
+ +
.
.
~3 y2a
.
+ -~
.
",/2b y2a
Cat 457
.
+ +
.
IG6 IH8
FPI45
.
+ +
IH4 IIA3
TI3
+ 4-, m c d i u m
titer;
+,
+ +
+ +
4- + +
+ ~ -
-
+ 4.
4 + +
-
+ * +
+ + +
-
4- + +
.
~- +
-
4- + +
+ ~ +
-
lov,' t i t e r ;
-,
+
negative.
219
TABLI.
3
lsolypicity and specificity of 14 m o n o c l o n a l antibodies produced v.'ith SP20 myeloma cells. Antibodies
Heavy
C.
chain
matis
tracho-
('. ])?lClgmOtli~lC
('. p s i t t a c i .
Avian strains
.
.
-
"tl
4
NI) ~ll
.
.
.
TI3
I.P145
(7at 457
A10
.
MPI
.
I.W679
K mouse
~
+
-~-
a
r~
* -
4 * t
t
~
.-
--
T
1
*
t
, + t
t-
..
~-
1
4-
t-
• + ÷.
~
a.
r
4-
t
4-
t
4-{"
I + ~-
•
• +
t
a. +
~--~ a-
-:-
t
4
i
+
t
4-
~ 4-
t
.~
~-
~
k-
4-
t
4-
~
t" 4
f
a
.
tt
f * ~ 4
k+
~-
t
4
4-
t-
a
~
~
~-
t
4-
.
,-
I
4-
4
t
,1
4-
~-
4
4
I
~
~-
-rl
We found good correlation with preceding work on serotyping of C. psittaci using polyclonal sera [11-14]. Twenty monoclonal antibodies had a crossspecificity between the ovine abortion strain A22 and another mammalian strain: K mouse• Eb [11], using polyclonal sera, had already described the high h o m o l o g y of antigenic structure between these two mammalian strains• On the other hand, we did not find any cross-specificity between two ovine strains which were pathogenically different (arthritis and abortion isolates). This is in accordance with De Long and Magee [15], who reported a monoclona[ antibody which was specific to an ovine abortion strain of C. psittaci and non-reactive with the ovine arthritis isolates. Finally, five monoclonal antibodies reacted to all C. psinaci strains and to the third species of Chlarnydia: C. pneumoniae (TWAR strain) but were non-reactive to C. trachomatis (LBI strain)• (7. pneumonia, the third species in the genus Chlarnydia on the basis of molecular biology [16, 17], was first classified as a C. psittaci strain because of the similarity in inclusion morphology [18]. We thought that this strain was also nearer to (7..psittaci than to (7.. trachomalts in antigenic structure. These results confirm the molecular biology and
t
-~
~ -
4
-t
4
t-
4
1 -
f-
t
~
1-
t
4
a-
-.
4"
l-
4-
t
4
"I-
T
+ 4-, High titer by the m i c r o i m n m n o f l u o r e s c e n c e technique; o, +, medium liter; + , l o w l i t c r ;
220
A22
44-
31 NI) 73 ~[1
.
.
•
"t2b
.
IO1. 2(.)7 .
Lolh *t2a "tl
.
M a m m a l i a n strains
1 B1
A5 AI7 AI9 A20 A21 A22 A23 B5 B6 BI2 BI3 BI6 B19 B22
.
•
4
, negative.
immunology studies; monoclonal antibodies appear to be a very good tool, capable of improving our knowledge of epidemiology and pathogenicity in
Chlamydia. References 111 Fuentes, V., l.efebvre, .I.I.., l e m a , F., Bissac, E. and Orfila, .I. (1985) l m m u n o l , l.ett. 10, 325. [21 Pakahashi, T., Takashima, 1. and Hashimolo, N. (1988) Microbioh l m m u n o l . 32, 251. [3] Seki, C., l a k a s h i m a , I., Arikav, a, J. and Hashirnoto, N (1988) .Ipn. J. Vet. Sci. 50, 383. [4] Fukushi, H., Nojiri, K. and Hirai, K. (1987).I. ( l i n . Microbiol. 25, 1978. 15] Toyofuku, H., l a k a s h i m a , 1., Arikawa, J. and Hashimoto, N. (1986) Microbiol. lmmunol. 30, 945. [6] Fuentes, V., Puy, H., Lefebvre, J. F. and Orfila, J. (1989) Microhios I.ett. 40, 55. [7] Fuentes, V., I-b, F. and Orfila, J. (1988) J. P,iol. Stand. 16, I. [8] Kohler, G. and Milstein, C. (1979) Nature 296, 495. [91 Wang, S. P. and Grays/on, .1. I. (1970) Am..I. O p h t h a h n o l . 70, 367. [10] Andersen, A. and Van Deusen, R. (1988) Infect. h n m u n . 56, 2075. [111 Eb, I..,Orfila,.I., Milon, A. and (ieral, M. I-.(1986) Ann. Inst. Pasteur Microbiol. 137 B, 77. [12] Perez-Martinez, J. A. and Storz, J. (1985) Infect. l m m u n . 50, 905.
[13] Schachter, .I., Banks, J., Sugg, N., Sung, M., Storz, J. and Meyer, K. E (1974) Infect. lmmun. 9, 92. [14] Schachter, J., Banks, J., Sugg, N., Sung, M., Storz, J. and Meyer, K. E (1975) Infect. lmmun. I1,904. [15] Delong, W. J. and Magee, W. E. (1986) Am. J. Vet. Res. 47, 1520.
[16] Cox, R. L., Kuo, C. C., Grayston, J. T. and Campbell, L. A. (1988) Int. J. Syst. Bacteriol. 38, 265. [17] Grayston, J. T., Kuo, C. C., Campbell, L. A. and Wang, S. P. (1989) Int. J. Syst. Bacteriol. 39, 88. [18] Grayston, J. T., Kuo, C. C., Wang, S. P. and Altman, J. (1986) New Engl. J. Med. 315, 161.
221
