Vetermary Immunology and Immunopathology, 25 (1990) 269-278 Elsewer Scmnce Pubhshers B V, Amsterdam - - Printed m The Netherlands

269

An A n t i g e n Capture E L I S A Test u s i n g M o n o c l o n a l A n t i b o d i e s for the D e t e c t i o n of Mycoplasma californicum in Milk H J BALL, D P MACKIE, D FINLAY, J McNAIR and D A POLLOCK

Vetermary Research Laboratortes, Stormont, Belfast BT4 3SD (Northern Ireland) (Accepted 7 December 1989)

ABSTRACT

Ball, H J , Mackm, D P , Fmlay, D , McNmr, J and Pollock, D A, 1990 An antigen capture ELISA test using monoclonal antlbodms for the detectmn of Mycoplasma cahforntcum m milk Vet Immunol Immunopathol, 25 269-278 The use of monoclonal antlbodms to detect Mycoplasma cahfornlcum was investigated m an antigen capture mmrotltre format The fmahsed test was highly specific and no cross-reactmns were detectable with any of the mastltxs assocmted mycoplasma or bactemal antigens tested Using a concentration step mvolwng centrffugatmn, the sensltlwty of the test could be improved from 105-107 to 10~-10 ~ colony forming umts per ml with pure broth cultures, and from 10v to at least l0 s colony forming umts per ml m milk samples from two expemmentally infected cows The antigen detected was partmlly identified by xmmunoblottmg, which demonstrated two polypept~des of 40 and 46 kD

INTRODUCTION

Mycoplasma cahforn~cum is a semous cause of bovine mastltlS, resulting m a rapid drop m milk ymld of infected glands (Mackm et al, 1982 ) The orgamsm was first isolated, charactemsed and named m Cahforma (Jasper, 1977, Jasper et al., 1981), and has since caused herd outbreaks m Europe (Mackle et al, 1982; Jurmanovel et al., 1983; PFutzner et al, 1986; Gibbs and Allan, 1986 ) M cahfornwum has been isolated from the jomts of a calf (Pfutzner et al, 1986), but no evidence of systemic spread has been observed m both naturally occurnng and experimental refection m Northern Ireland (Mackm et al, 1982, 1986) Low level excretion m the milk from latent refections or carrier ammals, as has been seen with M bovtgemtahum (Mackm and Ball, 1984), has not been observed wtth M cahforntcum Its Isolation from the gemtal tracts of small numbers of cows (Nakamura et al., 1977; Mackm et al, 1986) and bulls (Un0165-2427/90/$03 50

© 1990 Elsevmr Scmnce Pubhshers B V

270

H J BALL ET AL

gureanu et al, 1986, Ball et al, 1987a) indicates a poss:ble source for mammary gland mfectmn as well as a source for dlstnbutmn between ammals ELISA tests have been developed for the detection of antlbodms to some mastitls causing mycoplasmas in milk (Jurmanovd et al, 1986) and serum (Thomas et al, 1987) In addltmn ELISA methods using monoclonal antlbodms have been used to detect M bows antigen m milk (Boothby et al, 1986) and semen and preputial washings (Nmlsen et al, 1987) The present study was to investigate the use of monoclonal antlbodms to detect M cahforn~cum antigen m milk MATERIALS AND METHODS

Monoclonal antibodies BALB/c mine were lmmumsed with M cahforn~cum whole cell ant:gen, whmh consisted of washed cells, 50-100 fold concentrated from broth culture, disrupted by ultrasomcatlon Three days after the final mtrasplemc moculatmn, the spleen cells were fused with NSO myeloma cells (Galfre and Mflstem, 1981, Teh et al, 1984), and the hybndoma cells maintained m RPMI 1640 medmm supplemented w:th 20% gamma-globuhn free horse serum (Glbco) The culture flmds from acttvely growing hybndomas were screened by an ELISA test m m:crotltre plates (Dynatec) coated with the M cahforn~cum ~mmumsmg antigen Those producing h:gh ELISA readings were cloned twine by hm:tmg dflutmn and their monoclonal (MAb) antibody class determined by double lmmunodfffusmn against rabbit antlsera to mouse lmmunoglobuhn :sotypes (Nordic) Each MAb cell hne was rejected mtrapentoneally into a BALB/c mouse 4 days after the priming of the mouse with Freund's incomplete adjuvant (Mueller et al, 1986) The developed asotes fired was removed approximately 10 days later and stored at - 20 ° C ELISA The antigen capture ELISA was carrmd out m mmrotltre trays using 100 zl volumes of each reagent per well The optimum dllutton of each reagent was estabhshed by tltrat:on Capryhc ac:d purffied (McKmney and Parkmson, 1987) IgG fraction from the asotes fired of MAb 4H5 m 0.05 M carbonate buffer, pH 9 5 was used to coat the wells overmght at 4 °C The test samples were treated with detergent by incubating 1"2 dtlutlons of each m 1% Tween 80 m 0 01 Mphosphate buffered sahne (PBS), pH 7.2, at 37°C for 15 mm All further incubations were at 37°C and after each incubation stage, the wells were washed with five changes of 0 01 M PBS, pH 7.2, containing 0 05 % Tween 20 The stages following coating were as follows" the test sample for 2 h; the second purified MAb 5G6, whmh had been blotmylated (Hofmann et al., 1982 ), for 1 h, strepavldm-peroxldase (Sigma) for 1 h; and the substrate, whmh con-

AN ELISA TEST FOR THE DETECTION OF MYCOPLASMA CALIFORNICUMIN MILK

271

slsted of 0.4 mg o-phenylenedmmme dlhydrochlonde (Sigma)/ml and 0 4 pl/ ml (v/v) 30% H202 m 0.1 Mcltrate/phosphate buffer, pH 5.5, for 10 mm The substrate reactmn was stopped by the add~tmn of 50 ttl 2.5 M H2SO4 per well, and the absorbance was measured at wavelength 492 nm on an ELISA plate reader (Tltertek). Varmtmns of the above finahsed ELISA method included coating the mlcrot~tre wells with polyclonal rabbit serum rinsed to whole cell antigen, the use of different bmtmylated MAb's at the relevant stage and the use of combmatmns of bmtmylated MAb's

Experimental samples Milk samples collected from M cahforn~cum mastms herd outbreaks (Mackm et al, 1982, 1986) and from cows and ewes experimentally infected with M cahforn~cum, M bowgen~tahum, M canadense or ureaplasmas (Mackm and Ball, 1984, Ball and Mackie 1985, 1986; Ball et al, 1987a) and stored at - 70 ° C, were used m the initial standardlsatlon of the antigen capture ELISA Fresh milk samples from the commoner causes of bacterial mastltls caused by Staphylococcus aureus, Streptococcus spp. and Eschench~a coh were also examined Table 1 hsts the M cahfornicum and other mycoplasma broth cultures exammed All mycoplasmas, except M d~spar, were grown m PPLO broth (Oxold, Ball et al, 1987b) M d~spar was grown m Frns broth (Frns, 1975) To determine whether the senslt~vlty of the test could be ~mproved by concentratlon of the antigen by centnfugatlon, the following procedure was carned out with 1-day cultures of seven stratus of M cahforn~cum Ten-fold dllutmns m 20 ml volumes of sahne were centrifuged at 3000 rpm on a bench centrifuge for 20 mm The deposited cells were resuspended m 200-500 gl detergent solutmn and incubated at 37 ° C for 15 mm before testing TABLE 1 Mycoplasma b r o t h cultures examined by the antigen capture E L I S A test Specms

No of stratus

M cahfornlcum M bowgenttal~um M canadense M bows M alkalescens M bowrhm~s M arg~nm~ M verecundum M dtspar Acholeplasma la~dlawn

10 a 4 3 2 1 1 1 1 1 2

aStrams from N o r t h e r n Ireland, U S A , Czechoslovakia a n d East G e r m a n y

272

H J BALL ET AL

To assess the sensltlwty of the test with fresh milk samples, two quarters from each of two separate cows were expemmentally infected with M cahforn~curn M,lk from an uninfected quarter m each animal was used as a control Milk was collected twice a day at 09 30 and 16 00 h Following the estabhshment of a chronic mfectmn, with high milk cell levels ( > 107 cells/ml) and lowered milk ymld, each infected quarter was treated with m t r a m a m m a r y ant~bmtic Both quarters were treated following the morning and afternoon milkmg for 3 days, one with 1000 mg oxytetracychne and the other w~th 1000 mg tylosm per dose Milk samples were collected until the antigen was not detected by the antigen capture E L I S A test Each milk sample was tested as whole milk and, following centnfugatlon of a 20 ml volume, as separate cream, whey and cell samples

Mycoplasma t~tratmn The M cahforn~cum tltre of some samples was estimated by the transfer of measured volumes of 10-fold broth dflutmns to P P L O agar (Ball et a l , 1987b) After mcubatmn for 2-4 days at 37°C, the colony count was expressed as colony forming umts (cfu) per ml

Immunoblottmg After 2 days mcubatmn at 37 ° C, mycoplasma broth culture was centrifuged at 30 000 g for 30 m m and the concentrated cells washed three times with sahne The washed cells, contained in an ice bath, were somcated at 20 kilocycles per second for 3 × 1 m m intervals m an M S E 150W-ultrasomc d~smtegrator Crude membrane and cytoplasmm fractions were separated by centrffugatlon at 30 000 g for 30 mm, and the deposited membrane fraction washed three times with sahne S D S - P A G E was carried out w~th mycoplasma fractmns on 10% slab gels with 5% stacking gel Strips were removed for protein staining and the remainder of the gel apphed to a mtrocellulose sheet and blotted overmght at 17 m A / c m Strips ofmtrocellulose sheet from each fraction were blocked wlth 1% b o w n e serum albumin ,n 0 01 M P B S w~th 0 001 M E D T A and 0 5% Tween 80, and then incubated with dflutmns of the MAb's followed by rabbit ant~mouse IgG conjugated with horseradish peroxldase (Nordic) Each m c u b a t m n was at 37 °C and the strips were washed between reagents as descmbed above The peroxldase substrate used was 0 5 mg 3,3'-dmmmobenzld, ne tetrahydrochlonde ( S , g m a ) / m l m 0 02 M T n s / H C 1 buffer, p H 7 2, with 0 3 pl H202 (30% s o l u t m n ) / m l (v/v) RESULTS Twenty-s~x of the original 200 h y b n d o m a s actively secreted antibody to M

cahforn~cum antigen as determined by the E L I S A test. Five stable hnes were cloned, and all were found to be secreting antibody of the IgG1 lsotype

AN ELISA TEST FOR THE DETECTION OF MYCOPLASMACALIFORNICUMIN MILK

273

TABLE 2 S e n s l t l w t y of t h e a n t i g e n c a p t u r e E L I S A t e s t a p p h e d to f r a c t m n s of centrifuged b r o t h cultures

M cahformcum strata

162 390 358 MC547 102 St-6 M249

Broth culture tltre (cfu/ml)

D e t e c t m n tltre ( c f u / m l ) of a n t i g e n c a p t u r e E L I S A Whole broth

Centrifuged deposit

Supernatant

0 5 X 107 1 5×10 e 1 ×lO s 18X10 s 0 7 X 10 s 14×107 2 ×107

0 25 × 106 0 75×105 0 5 ×107 09 XIO s 0 35 × 106 0 7 ×105 1 ×107

0 25 × 105 0 75X103 0 5 ×105 09 ×10 ~ 0 35 X 10 ~ 07 XIO a 1 X10 ~

0 25 × lO s 0 75×10 s 0 5 ×107 0 9 ×107 0 35 X 107 0 7 ×107 1 ×107

TABLE 3 D e t e c t i o n of M cahformcum m t h e milk of e x p e r i m e n t a l l y infected m a m m a r y glands u s i n g t h e a n t i g e n c a p t u r e E L I S A t e s t Cow 1 w a s m o c u l a t e d w i t h 1 8 × 105 cfu a n d Cow 2 w i t h 3 8 × 10 v cfu D a y s p o s t - Cow no 1 lnoculum Mycoplasma Antigen capture E L I S A tltre m detection milk (cfu/ml) W h o l e Cells W h e y C r e a m milk

Mycoplasma Antigen capture E L I S A t~tre m detectmn milk (cfu/ml) W h o l e Cells W h e y C r e a m milk

lam p m 2am pm 3a m p m 4a m p m * 5am* pm* 6am* pm* 7am* pm

+ve (via b r o t h ) 5 X 10 3 13XlO 4 04×106 > 101° > 101° > 101° > 101° 3XlO 3 . .

.

.

.

.

4×10 s

.

.

. . + + + + -

. .

. .

. . -+ + + + -

1 X 10 3 13XlO e 09XlO 7 > 101° > 101° > 101° > 101° -

. -+ + + + + + +

.

.

+ + + + + + .

.

-

--

+

. .

-. .

+ . .

. .

Cow no 2

+ + + + -.

. .

. . . .

. . . .

. . . .

. . . .

.

.

. -+ + + + + + +

+ + + + + + ÷ -. .

. .

.

+ + + + + + + +

. . . .

* I n t r a m a m m a r y a n t l b l o t m t r e a t m e n t , Cow 1 w i t h o x y t e t r a c y c h n e a n d Cow 2 w i t h t y l o s m B o t h w i t h 1000 m g p e r dose

274

H J BALLET AL

;.6K iOK

Fig 1 (a) Coomassm bmlhant blue stained polypeptldes from sections of an SDS-PAGE slab gel ( 10% w/v acrylamlde ) of M cahforntcum membrane and soluble fractmns The molecular we:ght markers were measured with myosin (205 000), fl-galactosldase (116 000), phosphorylase B (97 400), bowne albumin (66 000), egg albumin (45 000) and carbomc anhydrase (29 000), and used to estimate the molecular weights of the arrowed polypeptldes

Clearest results were obtained by plates coated w~th purified MAb 4H5 and developedwlth blotmylated MAb 5G6 Positive reactions were obtainable with other combinations of MAb, including both coating and developmg w~th 4H5, but all were marginally less sensitive than the finally selected combination T~tratlon of strong poslt:ve reactmns showed a rapid fall off m act~wty at the end point, clearly separating poslt:ve reactions from the background level, which was typmally low (below 0 200 optmal umts) Any reading greater than twice the average background level was taken as a positive reaction Only strong pos~t:ves were detected following coating of the wells w~th polyclonal rabbit antiserum and the background level of activity was higher with

275

AN ELISA T E S T FOR T H E DETECTION OF MYCOPLASMA CALIFORNICUM IN MILK

2G2

4H5

5D(

2G2

4H5

51:)6

F r"

J

46K-il i 40K

(b)

l,

membrane

soluble

-I

Fig 1 (b) The remainderof the slab gel was blotted onto mtroeellulose Strips of the mtroeellulose corresponding to the membrane or soluble fractmns on the slab, were mcubated with MAbs and developedwith rabbit anti-mouseIgG conjugatedwith horseradishperoxldase The arrowedstamed bands correspondto the positron of those arrowed m Fig 1 (a) th~s coating. Combinations of blotmylated MAb dad not improve the sensitivity of the test. Broth cultures of all the M cahforn~cum stratus tested were clearly detected by the standardased antigen capture E L I S A Cross reactions were not detected with any of the other bovine mycoplasma broth cultures, or with any of the mastltm milk samples containing M bowgen~tahum, M canadense, bovine or ovme ureaplasmas, Staphylococcus aureus, Streptococcus agalactme, S dysagalact~ae, S ubens or E coh Concentrations of the M cahforn~cum cellular antigen by centnfugatlon dad increase the sensitwlty of the test, improving the level of detection m each broth by 10-100 fold (Table 2). Actwlty was, however, still detectable in the supernatant of the more concentrated dilutions, mdacatmg t h a t not all the activity was sedamentable under the condatlons used. In the two experimentally infected cows, the antigen was first detected m both ammals from the cell fractmn of the centrifuged milk only, when the mycoplasma tltre m the milk has risen to > 105 c f u / m l (Table 3) In both ammals the antigen was detectable from samples taken subsequently until after the start of antibmt~c treatment, even after the orgamsm could no longer be cultured The most pronounced immunoperoxldase actwlty of the blotted mtrocellulose strips was at one of two different rotes, and was present m both soluble and membrane fractmns {Fig. l b ) T h a t of 4H5 and two other MAb's was

276

H J BALL ET AL

eqmvalent to a strong band m the protein stained soluble mycoplasma fractmn, in the slab gel, of approximately 40 000 kD (Fig l a ) The staining of a band in the same positron m the membrane fraction was not so pronounced The activity m MAb 5G6 and one other MAb was again present in both membrane and cellular fractmns and equivalent to a faintly protein staining band m both, in the slab gel, at approximately 46 000 kD DISCUSSION An antigen capture E L I S A test has been developed for the rapid detection of M cahformcum in milk The test as currently used has excellent specificity, with no detectable cross reactions to any of the mastltls causing organisms tested Other E L I S A tests developed for demonstrating M cahformcum infection have concentrated on antibody detection (Jurmanovel et al, 1986, Thomas et al, 1987) Cross reactions with other mycoplasma antigens have been demonstrated, and the existence of specific M cahformcum antibody does not necessarily imply a current infection The specificity of the antigen capture E L I S A described in the present study is probably due to the use of MAb's for both capture and development stages, and is a welcome feature of mycoplasma serological tests, where cross reacting antigens are a well recognlsed problem (Kenny, 1977) The absence of any cross reaction in the test to M bov~gemtahum strains is of particular interest, not only because M bowgemtahum can be a cause of bovine mastltlS itself, but also because it shares an antigen with M cahformcum detectable by double lmmunochffuslon (H J. Ball, unpublished results) The sensitivity of the test can be improved by a concentration step to 10310 ~ c f u / m l with broth cultures Because of the rapid increase in the tltre of the organism in the milk of the experimentally infected animals, the sensitivity limit for the milk was not accurately measured, b u t was also Improved by concentratlon to at least 0 4 and 1 3 × 10 ~ cfu/ml. This level of sensitivity IS probably adequate for the detection of the majority of infected animals, since subclinical Infection with low level excretion is not recognlsed in disease with this organism The culture method used in this laboratory for screening milks for mycoplasma mastitis involves streaking milk samples (10/A) onto mycoplasma solid media This has a sensitivity of 102 cfu/pl and takes at least 2 days before mycoplasma colomes can be recognised in the midst of milk debris Serological identification of the mycoplasma species can often be further delayed by the need to subculture the primary isolate The screening of milk samples in a test such as the antigen-capture ELISA, has definite advantages over culture in speed and ability to handle large numbers The antigens involved in the test have only been characterlsed to a hmlted extent The two polypeptldes involved must be closely associated m the con-

ANELISATESTFORTHE DETECTIONOFMYCOPLASMACALIFORNICUMIN MILK

277

dltlons of the test, possibly belonging to the same protein. The larger amount of antigen of molecular weight 40 000 in the soluble rather than the membrane fraction, and the continued detection of the antigen m the supernatant of some centrifuged samples, m&cates that not all the antigen is strongly cell bound The fact that the MAb's used in the capture test were selected initially by their ELISA reaction to M cahforn~cum whole cell antigen, lmphes that the antigen detected was present m high levels in the whole cell antigen, and adsorbed well to the mmrotltre plate Whether a more sensitive antigen capture ELISA test for this mycoplasma could be developed by the selection of a different antigen, possibly purified by fractlonatmn, is open to speculation

REFERENCES Ball, H J and Mackm, D P , 1985 The ovme mammary gland as an experimental model to determine the virulence ofammal ureaplasmas J Hyg, 95 375-382 Ball, H J and Mackm, D P , 1986 Experimental production of bovine and owne mastlt~s with a Mycoplasmacanadense isolate Vet Rec, 118 72-73 Ball, H J , Logan, E F and Campbell, J N , 1987a Mycoplasma cahforn~cum m ewes as an experimental model for antlblotm treatment Epldemlol Infect, 98 369-378 Ball, H J , Logan, E F and Orr, W , 1987b Isolation of mycoplasmas from bowne semen in Northern Ireland Vet Rec, 121 322-324 Boothby, J T , Mueller, R , Jasper, D E and Thomas, C B , 1986 DetectmgMycoplasma bows m milk by enzyme-hnked lmmunosorbent assay, using monoclonal antlbodms Am J Vet Res, 47 1082-1084 Frns, N F , 1975 Some recommendations concerning primary isolation of Mycoplasma su~pneumonroe and Mycoplasma flocculare Nord Vet -Med, 27 337-339 Galfre, G and Mllsteln, C, 1981 Preparation of Monoclonal Antlbodms Strategms and Procedures Methods m Enzymology 73B Academic Press, London, pp 3-46 Gibbs, H A and Allan, E M , 1986 Mycoplasma mastltls outbreak Vet Rec, 118 647 Hofmann, K , Titus, G , Montlbeller, J and Finn, F M , 1982 Awdm binding of carboxyl-subst~tuted bmtm and analogues Bmchemlstry, 21 978-984 Jasper, D E , 1977 Mycoplasma and mycoplasmal mastltls J Am Vet Med Assoc, 170 11671172 Jasper, D E , Erno, H , Delhnger, J D and Chmstlansen, C, 1981 Int J Syst Bacterml, 31 339345 Jurmanov~i, K , H~ijkov~i,M and Vdvoda, J , 1983 Further ewdence of the involvement of Mycoplasma cahforn~cum m bowne mastltls in Europe Vet Rec, 112 608 Jurmanov~, K , H~jkov~, M and A~merov~i,D , 1986 The use of ELISA for detectmn of antlbodms against mycoplasmas and ureaplasmas m milk of mastltm cows Arch Exp Vet Med, Leipzig, 40 67-74 Kenny, G E , 1977 Antlgemc analysis of the Mycoplasmatates In D Hobson and K K Holmes (Editors), Nongonococcal Urethmtls and Related Infectmns Amerman Socmty for Mmroblology, Washington, DC, pp 376-382 Mackm, D P and Ball, H J , 1984 Pathogemc varmtmn of three stratus ofMycoplasma bowgen~tahum for the bowne mammary gland Vet Rec, 114 456-457 Mackle, D P , Ball, H J and Logan, E F , 1982 Isolatmn of Mycoplasma cahfornlcum from an outbreak ofbowne mastltm and the experimental reproductmn of the disease Vet Rec, 110 578-580

278

H J BALL ET AL

Mackm, D P , Ball, H J and Logan, E F , 1986 Mycoplasmacahforn~cummastltls m the dry dairy cow Vet Rec, 119 350--351 McKmney, M M and Parkmson, A, 1987 A s~mple, non-chromatographm procedure to purify lmmunoglobuhnsfrom serum and ascltes fired J Immunol Methods, 96 271-278 Mueller, U W , Hawes, C S and Jones, W R , 1986 Monoclonal antibody production by hybrldoma growth m Freund's adjuvant primed mine J Immunol Methods, 87 193-196 Nakamura, R M , Walt, M L and Bennett, R H , 1977 Studms of bovine gemtal tracts Mycoplasma isolations from dairy cows Therlogenology, 7 351-355 N~elsen, K H , Stewart, R B , Garcla, M M and Eaglesome, M D , 1987 Enzyme lmmunoassay for detection of Mycoplasma bows antigens m bull semen and preputial washings Vet Rec, 120 596-598 Pfutzner, H , Wehnert, Ch and Le~rer, R, 1986 Studms into udder virulence of Mycoplasma cahformcum Arch Exp Vet Med, Leipzig, 40 56-62 Teh, C -Z, Wong, E and Lee, C -Y G, 1984 Generation of monoclonal antlbodms to human gonadotrophm by a facile cloning procedure J Appl Blochem, 6 48-55 Thomas, C B , Jasper, D E , Boothby, J T and Delhnger, J D , 1987 Enzyme-hnked ~mmunosorbent assay for detection of Mycoplasma cahforntcum-speclfm antibody m bovine serum Opt~mlzatmn of assay determinants and control of serological cross-reactmns Am J Vet Res, 48 590-595 Ungureanu, C, Gngore, C, Iomta-Ionescu, F1 and Constantmescu, L, 1986 Frequency of mycoplasma m the semen of reproductmn bulls Arch Exp Vet Med, Leipzig, 40 82-87

An antigen capture ELISA test using monoclonal antibodies for the detection of Mycoplasma californicum in milk.

The use of monoclonal antibodies to detect Mycoplasma californicum was investigated in an antigen capture microtitre format. The finalized test was hi...
891KB Sizes 0 Downloads 0 Views