Rapid and simple identification of mycoplasmas by immunobinding

Journal oflmmunologicalMethods, 85 (1985) 257-267

257

Elsevier JIM03729

Rapid and Simple Identification of Mycoplasmas by Immunobinding H i t o s h i K o t a n i a n d G e r a r d J. M c G a r r i t y Departrnent of Microbiology, Institute for Medical Research, Copewood Street, Camden, NJ 08103, U.S.A. (Received 6 August 1985, accepted 4 September 1985)

A simple and rapid method of species identification of mycoplasmas by immunobinding assay is described. Small amounts of antigen of supernatant from cell cultures, broth cultures or clinical specimens were spotted onto nitrocellulose paper. This was followed by application of specific anti-mycoplasma antisera. After incubation, an enzyme-conjugated antiserum against the first antiserum was applied. A positive reaction was indicated by the development of intense blue color reaction when substrate was added. This method identified mycoplasma species with monoclonal and polyclonal antibodies. It detected 9.3 x 10 3-7.5 x 10 4 C F U / m l of organisms depending on mycoplasma species. For identification of mycoplasma, ureaplasma, acholeplasma and spiroplasma species, this assay is useful and rapid compared with other serological methods. In limited studies, the method correlated with microbiological assay of clinical specimens for Mvcoplasma pneumoniae. Key words: mycoplasma- immunobinding- nitrocellulose

Introduction Any technique that increases the overall efficiency of identification of mycoplasmas is of interest. A variety of techniques have been used for serological identification of mycoplasmas. These include growth inhibition (Clyde, 1964), metabolism inhibition (Ogata et al., 1979), epifluorescence of mycoplasmal colonies on agar (DelGiudice et al., 1966), immunofluorescence in cell cultures (DelGiudice and Hopps, 1977), ELISA (Tully et al., 1980), and immunoperoxidase (Hill, 1978; Polak-Vogelzang et al., 1978). Rapid detection and/or identification methods are useful. Taylor-Robinson has reviewed some of these and other criteria (1978). Blotting of DNA and proteins to nitrocellulose (NC) membrane filters from agarose or polyacrylamide gels have been widely used as analytical techniques (Towbin et al., 1979; Bowen et al., 1980). Dot blotting has been adapted for spotting protein antigens directly onto nitrocellulose paper (Hawkes et al., 1982). This method has been used for screening of monoclonal antibodies (Sternberg and Jeppesen, 1983). The objective of this study was to develop a rapid, simple and sensitive identifica-

0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

258 tion method for mycoplasmas using immunobinding assay on NC. Although the primary objective of these studies was to identify mycoplasmas isolated from cell cultures, the technique also has other potential applications, including clinical specimens.

Materials and Methods

Mycoplasma and media Ten mycoplasma species, 2 ureaplasma species, 1 acholeplasma species and 8 spiroplasma strains were used. Mycoplasma hyorhinis strain GDL, M. hominis strain PG21, M. arginini strain VA, M. salivarum strain VV, M. orale strain JS, M. pirum strain 70-159, M. pneumoniae strain FH and A. laidlawii strain M G were obtained from the American Type Culture Collection, Rockville, MD. M. fermentans strain H K is a triply cloned isolate from cell cultures in our laboratory. Ureaplasma urealyticum strain T960 (human) and ovine ureaplasma strain 1202 were obtained from departmental stocks. M. gallisepticum strain PG34, M. pulmonis strain PG31, Spiroplasma mirum strains SMCA, GT-48 and TP-2, S. sp. strains 277F, Y32, corn stunt spiroplasma (CSS) strain E275, AES-1 and MQ-4 were kindly provided by J.G. Tully of the National Institute of Allergy and Infectious Diseases, Bethesda, MD. Mycoplasmas and A. laidlawii were cultured in mycoplasma broth as described previously (McGarrity, 1982). Ureaplasma strains were cultured in T-broth as described (Ogata et al., 1979). Spiroplasma strains SMCA, GT-48, TP-2, 277F, Y32, AES-1 and MQ-4 were grown in SP-4 medium (Tully et al., 1977) and incubated at 37°C. CSS strain E275 was grown in M1D medium (Whitcomb, 1983) and incubated at 30°C. The number of viable mycoplasmas was determined by inoculation onto agar and recorded as the number of colony forming units (CFU)/ml). Cell cultures and cell culture mycoplasmas 3T6 mouse embryo fibroblasts grown in McCoy's medium + 10% fetal bovine serum (FBS) were infected separately with 8 mycoplasmas: M. hyorhinis, M. hominis, M. arginini, M. orale, M. salivarium, M. pirum, M. fermentans and A. laidlawii. To initiate infection, approximately 105 CFU of the respective mycoplasma species were inoculated into 4-6 h 3T6 cultures. The flasks were incubated at 37°C in 5% CO 2 in air for 4-5 days. The supernatants of these infected cultures were used as test antigens. Twenty cell cultures naturally infected with mycoplasmas were also used. These were submitted to our laboratory for mycoplasma assays by other institutions. The supernatants of these cell cultures were inoculated into 3T6 indicator cells with or without 6 methylpurine deoxyriboside (6MPDR) (MycoTech, BRL, Gaithersburg, MD) (McGarrity and Carson, 1982). This method has been used routinely in our laboratory for screening of mycoplasma infection in cell cultures. When 6MPDR assay revealed positive results, the supernatants were taken from cultures without 6MPDR (negative controls) and frozen at - 2 0 ° C before use (7 days or less).

259

Swab samples Swabs of oral cavities were collected from laboratory personnel. Clinical specimens were also obtained from patients with unknown respiratory infections. The swabs were immersed in 1.5 ml Tris-buffered saline (TBS, 50 mM Tris-HCl, 200 mM NaCI, pH 7.4) and vigorously agitated. The suspension was centrifuged at 15,000 × g for 10 rain in an Eppendorf centrifuge. The precipitates were resuspended in 0.2 ml TBS and used for immunobinding assay and for microbiological assay described previously (McGarrity, 1982). Polyclonal and monoclonal antibodies Polyclonal antisera against all strains were prepared in rabbits using the methods previously described (Ogata et al., 1979). Monoclonal antibodies against M. hyorhinis, M. arginini, M. salivarium, M. orale and A. laidlawii were also used (Buck et al., 1982). Immunobinding assay This assay procedure was modified from that described by Hawkes et al. (1982). Nitrocellulose (NC) paper (Millipore Corp., New Bedford, MA) was cut into squares, 2.5 × 2.5 cm, and placed in plastic petri dishes, diameter 6 cm. The NC paper was washed in distilled water for 5 min by agitation and left at room temperature to dry. The assay was performed at room temperature with agitation. Ten ~1 of the test specimens, supernatants of cell cultures, broth cultures or suspensions from oral cavity samples were applied as a spot and dried in air. Nine samples were placed on each NC sheet. To kill mycoplasmas, 10% buffered formalin was added to the NC sheet after the application of sample for 10 min and the sheet was then washed with TBS for 5 min. The NC sheet was pretreated to inactivate exogenous peroxidase by addition of 0.3% H202 in TBS for 10 min and washed with TBS for 5 min. Blocking solution, 10% normal horse serum, 0.02% Tween 20 in TBS, was added for 30 min. Polyclonal or monoclonal antibody in blocking solution was applied as a first antibody in a volume of 1 ml. The sheet was incubated for 30 rain at room temperature and washed 3 times with TBS, 3 min each wash. As a secondary antibody, peroxidase-labeled anti-rabbit IgG or anti-mouse IgG (Miles Laboratory, Naperville, IL) in blocking solution was applied for 30 rain, followed by 3 washings with TBS. Developing solution (3 mg 4-chloro-l-naphtol in 1 ml methanol, 5 ml TBS, 0.01% H 2 0 z) was added. A positive reaction consisted of a purple color which appeared in 5 to 15 s. After the reaction was completed, and before significant background developed, the sheet was washed with distilled water and dried. Growth inhibition test (GIT) and immunoperoxidase assay (IPA) Identification of mycoplasmas in infected cell cultures by immunobinding was compared with GIT and IPA. The techniques for GIT (Clyde, 1964) and IPA (Hill, 1978; Polak-Vogelzang, 1978) have been published.

260

Results Specificity and sensitivity of immunobinding assay. The specificity of the immunobinding assay was determined using 10 mycoplasma, 2 ureaplasma and 1 acholeplasma species. As a preliminary test, various blocking solutions, such as horse, fetal bovine, goat, lamb, pig sera, bovine serum albumin and Tween 20, were used. A combination of 10% horse serum and 0.02% Tween 20 in TBS provided best results without cross-reaction and non-specific staining. Incubation times of 30, 60, 90 and 120 rain were tested. Positive reactions, indicated as purple dots, were obtained by incubation times as short as 30 min at room temperature. When lower dilutions of conventional polyclonal antisera as a first antibody were used, nonspecific reactions appeared occasionally among some mycoplasma species as high background on nitrocellulose paper. However, when optimal concentrations of antibody such as 1:1000-36,000 were employed, such artifacts were virtually eliminated. In addition, even if some antisera were diluted 1:128,000, positive reactions still occurred. Table I shows specificity and sensitivity of this method. Both supernatants of 3T6 cells infected with mycoplasmas and broth cultures of mycoplasmas gave specific purple spots as homologous reactions using polyclonal antibody without cross-reactions. Some slight background appeared in a few cases. When monoclonal antibodies against A. laidlawii, M. hyorhinis, M. arginini, M. salivarium and M. orale were used for first antibodies, clear homologous reactions were obtained without non-specific reactions (Fig. 1).

TABLE 1 SENSITIVITY AND SPECIFICITY OF 1MMUNOBINDING MYCOPLASMA BROTH INOCULATED MYCOPLASMAS

Organism

A. laidlawii M. hyorhinis M. hominis M. arginini M. salivarium M. orale M. pirum M. fermentans M. pneumoniae M. pulmonis M. gallisepticum

Strain

ASSAY

ON

3T6

CELL

Source of mycoplasma ~

Sensitivity b

3T6 cells

(CFU/ml)

Mycoplasma broth

MG

+

+

1.1 X 1 0 4

GDL

+

+

1.0 ) 1 0 4

P G 21

+

+

2.5 × 10 4

VA

+

+

9.3 × l 0 S

VV

+

+

7.5 >( 1 0 4

JS

+

+

2.6 × 10 4

70-159

+

+

ND ~

HK FH

+ ND

+ +

1.5 × 10 4 2.2 × 10 4

P G 34

ND

+

ND

P G 31

ND

+

1.0 X 1 0 4

AND

"~No cross-reaction occurred among these strains using polyclonal antibodies against all species and m o n o c l o n a l antibodies against A. laidlawii, M. hyorhinis, M. arginini, M. salwariurn and M. orale. b Organisms were cultured in mycoplasma broth. Not done.

261

A 1

4

2

.J

lx

1

4

t:

8

2

5

0

369.3 .",X _ -- .^_ c

69 __ __ ".__---D

"_ _.

14

147 2

5

3

69

8

7

2

5

8

3

6

9

Fig. 1. Immunobinding assay on cell culture samples using polyclonal and monoclonal antibodies. Ten ~1 samples from supernatants from mycoplasmas infected 3T6 cells were dotted onto NC paper. Samples were A. Inrdlrwii (1) M. hominis (2) M. orale (3). M. Inwrhinis (4). M. argmini (5). M. saliuarium (6). M. pirum (7) M. fermenmn~ (8) and McCoy’s medium (9), with 10% fetal bovine serum. Polyclonal antibodies against M. hyorhinis (A) and A. &d/awir (B) were used. Monoclonal antibodies against M. hvorhrnrs (C) and A. laidlawri (D) were also employed for comparison.

To determine the sensitivity of this method, lo-fold serial dilutions of broth cultures of each species were applied to the same NC paper. Fig. 2 shows representative results. The end-points of positive detection, listed in Table I, ranged from 9.3 X lo1 to 7.5 X lo4 CFU/ml, rendering this method as sensitive as many other identification procedures.

Fig. 2. Sensitivity of immunobinding assay. Ten-fold serial dilutions of M. arginini broth culture were applied to NC paper. Samples l-8 contained 10’. 106, 105. 104. lo’, 102. lo’, < 10’ CCU/ml organisms respectively. Sample 9 was mycoplasma medium.

262

Identification of mycoplasmas in naturally infected cell cultures Cell culture samples were used for identification of mycoplasma species (Table II). Of these, 16 samples were infected with a single mycoplasma species. M. arginini, M. fermentans, M. hyorhinis and M. orale infected 4, 2, 8 and 2 samples, respectively. This technique also detected mixed infections with mycoplasmas. Sample 456 contained A. laidlawii and M. hyorhinis. Sample 225 had M. arginini and M. fermentans. Sample 8074 had M. pneumoniae and M. orale. Sample 230 had M. arginini, M. fermentans and M. orale. Fig. 3 shows NC sheets used to identify mycoplasmas isolated from cell cultures. The results of immunobinding assays always correlated with results of growth inhibition tests and immunoperoxidase

T A B L E II IDENTIFICATION OF MYCOPLASMA T U R E S BY I M M U N O B I N D I N G A S S A Y

SPECIES

IN N A T U R A L L Y

Lab

Identification m e t h o d

sample ~

Growth

Immunoperoxidase

Immunobinding

INFECTED

CELL CUL-

Organism

inhibition test

test

assay

1 452

+

+

+

1 453 1 631 1 533 1 532

+ + ND ND h

+ + + +

+ + + +

229 1 472 1 501

+ NA ~ NA

ND + +

+ + +

1 502 1 515 104 105 106 107 1 603 1 902

NA NA NA NA NA NA + +

+ + ND ND ND ND + ND

+ + + + + + + +

456

+

ND

+

225

+

ND

+

8 074

+

+

+

230

+

ND

+

M. arginini M. arginini M. arginini M. arginini M. fermentans M. fermentans M. hvorhinis M. hyorhinis M. hyorhinis M. hyorhinis M. hyorhinis M. hyorhinis M. hvorhinis M. t~,orhinis M. orale M. orale A. laidlaw# and M. hyorhinis M. arginini a n d M. fermentans M. pneumoniae and M. orale M. arginini M. fermentans a n d M. orale

Broth cultures of 229, 225 a n d 230 were used. S u p e r n a t a n t s of infected 3T6 cells were used for othe, samples. h N o t done. N o t applicable. T h e strain was non-cultivable.

263

B

A

C;

1

4

7

1

4

7

1

4

7

2

5

8

2

5

8

2

5

8

3

6

9

3

6

9

3

6

9

E

D 1

4

7

2

5

8

3

6

9

1

4

7

2

5

8

3

6

9

Fig. 3. Immunobinding assay on samples from mycoplasma infected cell cultures. Cell culture samples tested in Table iI, 1: 1533, 2: 1501, 3: 1502, 4: 104, 5: 456, 6: 1532, 7: 225, 8 : 1 9 0 2 and 9: McCoy's medium, were dotted onto each NC paper. Polyclonal antibodies against M. arginini (A), M. hyorhinis (B), A. laidlawii (C), M. fermentans (D) and M. orale (E) were used.

assays. All M. hyorhinis isolates failed to grow in both broth and agar media. The identification of 2 or 3 mycoplasmas from cell cultures by immunobinding was confirmed by growth inhibition, epi-fluorescence or immunoperoxidase assays. To date, the immunobinding assay has been used to successfully identify mycoplasmas isolated from 77 different cell cultures. Mycoplasma free cell cultures, D M E M + 10% FBS and undiluted FBS had no cross-reactions to any of the 10 mycoplasma species tested. In broth cultures of mycoplasmas, old or dead organisms were also identified by this method. Cultures as old as 2 months have been identified with this technique.

Comparison of spiroplasmas by immunobinding assay Eight spiroplasma strains were studied for serological comparisons (Table III). Corn stunt spiroplasma strain E275 and 277F strain reacted with each other, suggesting that these strains belonged to the same serogroup I as recognized previously (Tully et al., 1980). S. mirum strains SMCA, GT-48 and TP-2 were identical antigenically. Strain Y32 reacted with only homologous antiserum. Strains AES-1 and MQ-4 did not react with the antisera used. These results suggest that immunobinding ~issay can be useful for serotyping spiroplasmas.

Immunobinding of ureaplasmas The immunobinding assay was performed on Ureaplasma urealyticum strain T960

264

TABLE

Ill

COMPARISON

OF EIGHT

SPIROPLASMA

Serogroup

Species or strain a

I-3

Corn stunt

1-4

277F

V

S. m i r u m

STRAINS

BY IMMUNOBINDING

ASSAY

Antiserum E275

277F

+ +

spiroplasma: E275

SMCA

GT-48

TP-2

+

-

-

-

+

-

-

Y32

-

SMCA

-

-

+

+

+

GT-48

-

-

+

+

+

-

TP-2

-

-

+

+,

+

-

VI

Y32

.

.

X

AES-1

.

.

XI

MQ-4

.

" S t r a i n E 2 7 5 w a s c u l t u r e d in M I D

.

. .

.

medium;

. .

+ .

.

.

.

.

a ll o t h e r s t r a i n s w e r e c u l t u r e d in S P - 4 m e d i u m .

(serotype VIII) and ovine strain 1202. Weak cross-reactions occurred. Rabbit antisera prepared against T960 and 1202 strains had titers of 64,000 and 80,000 respectively in the immunobinding assay.

TABLE

IV

COMPARISON MAN

ORAL

OF MICROBIOLOGICAL

CULTURE

AND

IMMUNOBINDING

ASSAY

SPECIMENS

Sample ~

M. orale

M. salivarium

MA b l

IBA ~

MA

.

.

.

.

M. p n e u m o n i a e IBA

.

.

2

.

.

3

+

+

+

+

4

+

+

+

+

5

+

+

+

+

6

.

7

.

8

.

.

. .

.

. .

.

-

+

+

.

. .

IBA

.

.

.

MA

.

.

.

.

.

9

+

+

+

+

+

+

10

+

+

+

+

+

+

+

-

-

+

+

11

-

12

.

13

.

14

-

-

.

+

. .

. .

-

. .

+

Samples 1-8: apparently healthy persons. Samples 9-14: h Microbiological assay. lmmunobinding

assay.

.

. +

patients with respiratory symptoms.

ON

HU-

265

Identification of mycoplasmas from oral swab samples Oral swab samples were taken from 8 apparently healthy persons and from 6 patients with respiratory infections. Table IV shows the results of microbiological culture and immunobinding assays. M. orale and M. salivarium were identified from 5 and 7 samples respectively by both methods; M. pneumoniae was detected in 4 specimens by both methods. The time required for M. pneumoniae by culture was 12 17 days. Immunobinding assay results were available in 3 h.

Discussion

Different methods have been used for identification of mycoplasmas (Clyde, 1964; DelGiudice et al., 1966; DelGiudice and Hopps, 1977: Polak-Vogelzang et al., 1978; Tully et al., 1980). Growth inhibition tests and epi-immunofluorescence have been frequently used to identify mycoplasmas isolated from in cell cultures (McGarrity, 1982). Although these methods are simple to perform, they have a disadvantage. In recent surveys, the majority of M. hyorhinis isolates did not grow in mycoplasma broth and agar media (DelGiudice and Hopps, 1977: McGarrity, 1982). As an alternative method, cell culture immunofluorescence and immunoperoxidase tests have been used. These methods require cell cultures grown on coverslips and sometimes indicator cells. This may require an additional 3-4 days to identify the organisms. The identification method described here is rapid and simple. This was based on the application of antigens without any kind of pretreatment as a spot to NC paper. Immunobinding assay used in this study employed enzymatically labeled IgG. For identification of cell culture mycoplasmas, small amounts of supernatants were applied onto nitrocellulose paper. The complete immunobinding assay to identify mycoplasma species is performed in 3 4 h with high specificity and sensitivity. When optimal titer of antibody was used for primary antiserum, no untoward cross-reactions appeared among the species of mycoplasmas studied. Monoclonal antibodies also demonstrated clear reactions. The IBA was also used successfully in aging or dead cultures of mycoplasmas, a distinct advantage over some other techniques. Mycoplasma concentrations in the supernatants of infected cell cultures are usually 107-108 C F U / m l (McGarrity, 1982). Results of the present study show the immunobinding assay can detect approximately 104 C F U / m l of organisms, suggesting that the number of organisms in supernatant is enough to detect antigen. In our laboratory, microbiological assay (McGarrity, 1982), DNA fluorochrome staining (McGarrity, 1982) and 6MPDR assay (McGarrity and Carson, 1982) are routinely employed to detect mycoplasma infection in cell cultures. When the 6MPDR assay demonstrated positive cytotoxicity results, indicative of mycoplasma infection, 10/L1 supernatant of the positive well was taken as an antigen. Identification can be done the same day. For certain specimens, the amount of antigen, if necessary, may be adjusted by centrifugation or other means. The immunobinding technique has been used for screening production of m o n o -

266 clonal antibodies from h y b r i d o m a cells (Hawkes et al., 1982; Sternberg and J e p p e sen, 1983). Hsu has also applied this technique for detection of t o b a c c o mosic virus in leaf extracts using gold-labeled anti-rabbit I gG as a second a n t i b o d y (1984). It detected 1 - 5 pg of protein with high specificity. T h e technique can have a wide range of applications in the detection and identification of m y c o p l a s m a s from a variety of specimens. It has been applied directly to m y c o p l a s m a colonies on agar (manuscript in preparation). In addition to cell cultures, sera and specimens such as those from the respiratory and urogenital tracts of h u m a n s and animals, infected plants and insects and other vertebrates could be assayed. In our laboratory, the assay approaches the E L I S A in sensitivity and specificity. At the same time, it can be d o n e easier in pre-cut pieces of N C p ap er of a size that is d e t e r m i n e d by the nature of the test. Because of its overall ease and quickness, the i m m u n o b i n d i n g assay described here has potential application in the clinical laboratory to detect p a t h o g e n i c m y c o p l a s m a s and perhaps other microorganisms.

Acknowledgements These studies were supported by G r a n t AI-15748 from N I H . Th e authors thank Lynne A n d e r s o n and Judi S a r a m a for technical assistance. We thank J o a n n e M c C a f f r e y of G a r d e n State Hospital and Jack Goldstein, D.O. for supplying clinical specimens.

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267 Towbin, H., T. Staehelin and J. Gordon, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 4350. Tully, J.G., R.F. Whitcomb, H.F. Clark and D.L. Williamson, 1977, Science 195, 892. Tully, J.G., D.L. Rose, O. Garcia-Jurado, J.C. Vignault, C. Saillard, J.M. Bov6, R.E. McCoy and D.L. Williamson, 1980, Curr. Microbiol. 3, 369. Whitcomb, R.F., 1983, in: Methods in Mycoplasmology, Vol. I, eds. R. Razin and J.G. Tully (Academic Press, New York) p. 147.