Veterinary Microbiology, 20 ( 1989 ) 155-163 155 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

Use of Hydrophobic Cloths as Ant ibody Adsorbents for E n z y m e Immunoassay: Detec t ion of Brucella Ant igens

BURTON W. BLAIS 1, HIROSHI YAMAZAKP* and C H A R L O T T E E. RIGBY'-'

'Department of Biology, Carleton University, Ottawa, Ont. KIS 5B6 (Canada) -'Agriculture Canada Animal Diseases Research Institute, P.O. Box 11300, Station H, Nepean, Ont. K2H 8P9 (Canada)

(Accepted for publication 12 December 1988)

ABSTRACT

Blais, B.W., Yamazaki, H. and Rigby, C.E., 1989. Use of hydrophobic cloths as antibody adsor- bents for enzyme immunoassay: detection of Brucella antigens. Vet. Microbiol., 20: 155-163.

A cloth-ELISA (C-ELISA) antigen capture assay for the detection ofBrucella melitensis and B. abortus was developed. Segments (6-mm squares) of hydrophobic polyester cloth coated with diluted serum from a B. abortus-infected cow were incubated with saline suspensions of heat- killed Brucella cells, or with cultures of bovine or sheep blood or bovine tissue homogenates that had been inoculated with B. abortus or B. melitensis added to trypticase soy broth (TSB) and incubated for 2 3 days. The captured antigen was detected by a bovine anti-BruceUa antibody- horseradish peroxidase conjugate system. The enrichment culture technique detected as few as three Brucella colony-forming units (c.f.u.) in 0.5 ml of bovine blood and was positive in cultures in which the Brucella concentration had reached 3 × 106 c.f.u, ml ~ (after 2 or 3 days incubation ). The combined enr ichment-c loth-ELISA method gave complete correlation with cultural isolation and results were available 3 days before colonies appeared in conventional culture. Hydrophobic cloths have potential use in diagnostic procedures since they provide simple, rapid and economical assays.

I N T R O D U C T I O N

The definitive diagnosis of infectious diseases often involves the detection of pathogens or their antigens in clinical specimens. Several methods have been developed for the rapid detection of Brucella antigens in specimens, in- cluding a coaggulation test using coloured Staphylococcus aureus (Batra et al., 1987), agar gel precipitation and counter-immunoelectrophoresis (Chand et al., 1987) and an avidin-peroxidase complex immunoenzymatic staining tech-

*Author to whom correspondence should be addressed.

0378-1135/89/$03.50 © 1989 Elsevier Science Publishers B.V.

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nique (Meador et al., 1986). Alternatively, enzyme-linked immunosorbent as- say (ELISA) tests, which are increasingly popular for the detection of micro- bial antigens, may be suitable for the rapid and economic detection of Brucella antigens in clinical specimens (Perera et al., 1983). The simplest form of such a capture assay uses specific antibody immobilized on a solid phase to capture an antigen which then specifically binds an enzyme-conjugated antibody that produces a colour change in a suitable substrate. Capture antibody can be im- mobilized by covalent binding, e.g., by glutaraldehyde t rea tment of HCl-acti- vated nylon (Perera et al., 1983) or cyanogen-activated polyhydroxy supports (Yolken, 1982). Equally effective, the simplest and most economical method is simple adsorption to: (1) non-porous surfaces (e.g., microtitre plates, tubes or beads made of polystyrene, polyvinyl chloride or polymethacrylate); (2) microporous membranes made of nitrocellulose or nylon, or (3) macroporous cloths of hydrophobic synthetic fibres such as polyester. Such cloths are readily available and economical, offer a larger surface area than non-porous solid phases for binding immunoreactants , accommodate a larger volume of sample than microporous solid phases per unit area and are easily washed.

In this paper, we describe the development and preliminary evaluation of a cloth ELISA (C-ELISA) test for the detection of Brucella antigens, based on the use of polyester cloth as the solid phase. The C-ELISA was combined with broth enrichment for the detection of B. abortus or B. melitensis inoculated into blood or tissue homogenates.

M A T E R I A L S AND M E T H O D S

Antigens

B. abortus biovar 1 strain 413 (standard diagnostic antigen) was prepared at the Animal Diseases Research Insti tute (A.D.R.I.) and cells were counted in a Pe t rof f -Hauser chamber. B. abortus biovar 1 strain 807 (a field isolate) and B. melitensis strain 16M were from the A.D.R.I. culture collection. BruceUa cultures were grown on tryptose agar with 5% bovine serum and 1% glucose at 37°C in 10% CO2, and harvested in saline.

Antisera

Anti-Brucella sera DC950 and 36398N-2 were from adult cows with chronic brucellosis. Serum DC950 had titres of 1280 by complement fixation and 3200 by the standard tube agglutination test, whereas serum 36398N-2 had titres of 5120 and 3200, respectively. Both sera were tested as sources of capture anti- body for the C-ELISA. Affinity purified anti-Brucella antibody from serum DC950 (APDC950) was prepared by the acid technique of Read et al. (1974),

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using B. abortus 413 cells. Protein was determined by the method of Lowry et al. (1951).

Horseradish peroxidase ( H R P ) (Sigma No. P8385) was oxidized with per- iodate and conjugated to APDC950 antibody as described by Nakane and Ka- waoi (1974). The H R P - a n t i b o d y conjugate ( H R P C ) was stored in 0.01 M phosphate-buffered (pH 7.2) 0.85% NaC1 (PBS) at - 2 0 ° C . For use, it was diluted 1:1000 in PBS containing 0.05% Tween 20 (PBST) .

Coating

Polyester non-woven cloth (DuPont , Sontara 8100) was cut into 6 X 6-mm squares. Each square was placed in a covered plastic Petri dish and incubated with 50 ,ul of serum diluted 1:100 in PBS, then heated in a 75°C water bath for 10 min, maintained for 6-16 h at room temperature, then thoroughly washed with 5 ml PBST. This t rea tment not only coated the cloth with capture anti- body specific for Brucella, but also covered any non-specific binding sites on the cloth which might otherwise interact with HRPC. The antibody-coated cloth squares (ACS) were stored in PBS at 4 ° C. Other materials tested as solid phases in the ELISA included polypropylene cloth (Aldrich Chemical Co., No. Z10425-6), woven polyester cloth (Bouclair Textiles), woven nylon 6/6 cloth (Conval-Aid, Ottawa), polyethylene filter (Fisher Scientific, 1.5 mm thick- hess), analytical paper (Schleicher and Schuell, No. 740-E), cellulose nitrate membrane (Schleicher and Schuell, No. 33172) and cellulose acetate mere- brane (Gelman, No. 60172). These materials were cut into 6 X 6-ram squares and each segment was coated with 50 i~1 of pre-heated serum DC950 diluted in PBS. They were then incubated and washed with P B S T as above.

Tube C-ELISA test

Thirty microlitres of test sample were pipet ted onto each ACS in a Petri dish and incubated for 30 min at room temperature, then the ACS were placed on a disposable diaper and washed five times dropwise with ~ 0.3 ml of PBST. The cloths were returned to the Petri dish and one drop (30 ]xl) of H R P C was placed on each. After 30 min incubation at room temperature, the cloths were washed as above then placed in a 16 X 100-mm glass tube containing 0.5 ml indicator-substrate [0.05 M sodium citrate (pH 4.5) containing 10 mM 2-2'- azino-bis- (3-ethylbenzthiazoline sulphonic acid) (ABTS) and 0.5 mM H,~O~ ] and incubated for 30 min at room temperature. The reaction was stopped by the addition of 0.5 ml 0.1 M NaF and the absorbence at 414 nm was determined.

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Enrichment-C-ELISA rapid test

Enrichment Twenty culture-negative tissues were tested by a combined enrichment-C-

ELISA method. To 4.5 ml of trypticase soy broth (TSB) was added 0.5 ml of' tissue homogenate, and 1 ml was tested in the C-ELISA after 48 and 72 h incubation at 37 ° C ( see below). The tissues tested included two lumbar lymph node specimens, three spleens, two uteri, three supramammary lymph nodes, six prescapular lymph nodes, one retropharyngeal lymph node, two udders and one foetal spleen, all from different cattle. These tissues had been homogenized in a stomacher and cultured for B. abortus. All were culture negative.

Simulated samples were prepared by inoculating 10/~l of saline or of serial dilutions orB. melitensis 16M of B. abortus 807 in PBS into 0.5 ml of sheep or bovine blood, or tissue homogenate. The numbers inoculated were confirmed by viable counts. The inoculated suspensions were added to 4.5 ml of TSB and incubated at 37 ~C. After 48 and 72 h, 1 ml of culture was transferred to a 16 X 100-mm glass tube and a sample removed for viable counts. The tubes were heated at 60 ° C for 90 rain to kill the brucellae and inactivate blood per- oxidase(s), then cooled for 2 h before testing by C-ELISA. Positive controls contained 0.1 ml of blood and 10 ul of a heat-killed B. melitensis suspension (1 X l0 T c.f.u. ) added to 0.9 ml of TSB; the negative control contained 0.1 ml of blood in 0.9 ml of TSB. These were heated then cooled as above.

Rapid test An ACS was placed in each heated specimen of enrichment culture and in-

cubated for 45 min at 37 ° C for antigen capture, then placed on a piece of screen over a plastic container, washed with PBST (a total of 4 ml per cloth) and briefly blotted. One drop ( ~ 50 ~1) of HRPC was then added to each cloth. After 5 min incubation at room temperature, the cloths were washed with PBST as above and one drop ( ~ 50/L1) of ABTS substrate-indicator was added. Strong positive reactions produced a colour change to dark green (A4H > 0.8) within 2-3 min and reactions were recorded after 5-10 min. Negative reactions pro- duced little or no colour change (A414 < 0.2 ) within a 10-min incubation period.

RESULTS

Coating polyester cloth with antiserum

Heating the antiserum at 75°C for 10 rain prior to coating the cloth in- creased the C-ELISA signal using serum DC950 at a dilution of' 1:1000 and a substantial improvement in the C-ELISA signal was obtained using pre-heated serum 36398N-2 throughout at all dilutions tested (Fig. 1 ). This suggests that the heat t reatment increased immunoglobulin binding to the polyester cloth

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DILUTION

Fig. 1. Effects of heat t rea t ing ant i -Brucel la coating sera DC950 ( A, A ) and 36398N-2 ( O, O ) on the C-ELISA. Diluted sera were heated at 75 ~ C for 10 min ( A, O ) or left at room temperature (A , • ) . Polyester cloth segments (6-mm squares) were then coated with the sera (50ltl per cloth). The test ant igen was B. abortus 413 (1.3 X 10 ~; cells per cloth). The C-ELISA was per- formed as described in Materials and methods, and the cloths were incubated in ABTS indicator- substrate solution for 30 min and A414 recorded. Points are plot ted as mean A , , value _+ s tandard error ( n = 3 ) .

surface using serum 36398N-2 and to a lesser extent with serum DC950. The figure also shows that the coating sera can be diluted to at least 1" 1000 and still generate a detectable signal in the C-ELISA.

Comparison of various porous supports

A variety of porous materials were examined as supports for the tube C- ELISA test (Table 1 ). The test antigen was 3 × 105 B. abortus 413 cells applied per segment. Cellulose nitrate and cellulose acetate membranes could not be washed effectively between the reaction steps using the absorbent pad. When

T A B L E 1

Comparison of signals of the C-ELISA performed on various porous supports

Support Absorbence A, ,4"

Wi th B. abortus cells Wi thout B. abortus cells

Polypropylene filter cloth Cellulose ni t ra te membrane Cellulose acetate membrane Analytical filter paper Polyethylene filter Woven nylon 6 /6 cloth Woven polyester cloth Non-woven polyester cloth

1.20_+.0.02 0.68_+0.02 0.55_+0.02 1.10_+0.01 1.20_+0.02 0.86 _+ 0.02 2.21 _+0.02 2.10_+0.02

0.02 + 0.00 0.50+0.01 0.29_+0.02 1.06 + 0.03 0.18±0.01 0.02 + 0.00 0.06_+0.02 0.03 + 0.00

~'Mean ± s tandard error (n --3 ).

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washed with 5 ml P B S T using a vacuum filtration apparatus, they still exhib- ited high backgrounds. The filter paper similarly suffered high backgrounds. Woven nylon cloth gave low backgrounds, but the edges tended to ravel during the washing and handling. The highest signals were obtained using woven and non-woven polyester cloths, but these were not easily wettable and care was necessary to ensure uniform wetting of the cloths. Also, at low antigen concen- trations (3 × 104 cells per cloth), polypropylene and polyester cloths gave al- most identical signals in the C-ELISA (data not shown). The antibody-coated polypropylene and polyester cloths required a minimum of 30 min incubation for opt imum capture of B. abortus cells and for reaction with HRPC.

Detection of Brucella in enrichment broth by C-ELISA

B. melitensis Fresh sheep blood was inoculated with 10/A ofB. melitensis 16 M calculated

to contain 3000, 300, 30 or 3 cells (from viable counts) . After 48 and 72 h incubation in TSB, the cultures were tested by C-ELISA and viable counts were performed. Table 2 shows that with the combined enr ichment-C-ELISA technique, as few as 3 c.f.u, per 0.5 ml of sheep blood specimen were detected after 72 h incubation. Enrichment culture was necessary to allow multiplica- tion of brucellae to a minimum of ~ 1 × 10 ~ cells ml - 1 for detection by C-ELISA.

B. abortus The 20 Brucella-negative tissue homogenates tested by the enrichment-C-

ELISA rapid test were all negative after 48 and 72 h incubation, even though six were grossly contaminated with Gram-positive bacteria. Fourteen of these

T A B L E 2

De tec t i on by e n r i c h m e n t - C - E L I S A a n d cu l tu re o f B. melitensis in m o c k - i n f e c t e d sheep blood ~

No. of c.f.u, i nocu la t ed ~ 48 h i n c u b a t i o n 72 h i n c u b a t i o n

C - E L I S N ' Cu l tu re d C -ELISA" Cul tu re 'l

3000 + 6.5 + + 9.0 300 - 5.5 + + 9.0 30 - 5.0 + + 9.0 3 - 4.0 + + 9.0 0 (cont ro l ) - N D - N D

~'0.5 ml of sheep blood was i nocu la t ed w i t h 10/21 s u s p e n s i o n , 4.5 ml of T S B were added a n d t he cu l tu res i n c u b a t e d a t 37 ° C. %.f.u. = c o l o n y - f o r m i n g un i t s , ca lcu la ted f rom viable coun t s . ~+ , weak colour r eac t ion (0.2<A4~4 < 0 . 8 ) ; + + , s t r o n g colour r eac t ion (A414>0.8); - , no re- ac t ion (A414 < 0.2). a L o g ~ c.f.u, ml 1; ND, n o t d e t e r m i n e d .

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t i s s u e s a n d a Brucella-negative b o v i n e b l o o d s p e c i m e n w e r e i n o c u l a t e d w i t h 10

~l o f B. abortus 807 c a l c u l a t e d to c o n t a i n 55, 30, 5, 3 o r 1 c.f .u. ( f r o m v i a b l e

c o u n t s ), a n d i n c u b a t e d in T S B for 48 a n d 72 h. T h e s e w e r e t e s t e d b y C - E L I S A a n d v i a b l e c o u n t s w e r e p e r f o r m e d as a b o v e . N o b r u c e l l a e w e r e d e t e c t e d b y C- E L I S A or r e c o v e r e d o n c u l t u r e p l a t e s f r o m e i g h t o f t h e 14 t i s s u e s i n o c u l a t e d w i t h < 30 c.f .u. , p o s s i b l y b e c a u s e o f a n t i b a c t e r i a l s u b s t a n c e s in t h e t i s s u e s t h a t i n h i b i t e d s u r v i v a l a n d m u l t i p l i c a t i o n o f t h e b r u c e l l a e . T h e s e e i g h t t i s s u e s in-

c l u d e d o n e l u m b a r l y m p h n o d e , t w o s p l e e n s , o n e u t e r u s , one u d d e r a n d t h r e e s u p r a m a m m a r y l y m p h n o d e s . T a b l e 3 s u m m a r i z e s t h e r e s u l t s for t h e s ix p o s - i t i ve t i s s u e s a n d t h e b o v i n e b l o o d s p e c i m e n . A l l c u l t u r e - p o s i t i v e s a m p l e s w e r e a l so C - E L I S A p o s i t i v e ( t h e r e w a s c o m p l e t e c o r r e l a t i o n ) . T h e c o m b i n e d e n - r i c h m e n t - C - E L I S A t e c h n i q u e d e t e c t e d as few a s 3 c.f .u, p e r 0 . 5 - m l s p e c i m e n in b l o o d , o n e s p l e e n s a m p l e a n d t h r e e o f t h e f o u r l y m p h n o d e h o m o g e n a t e s

TABLE 3

Detection by enrichment-C-ELISA and culture of B. abortus inoculated into bovine blood or tissue homogenates.

Tissue No. of c.f.u. 48 h incubation 72 h incubation inoculated a

C-ELISA ~' Culture" C-ELISA t' Culture'

Blood 30 3 1

Spleen 3 Prescapular 55 Lymph Node 1 5

Prescapular 3

Lymph Node 2 1

Prescapular 3

Lymph Node 3 1

Prescapular 3

Lymph Node 4 1

Supramammary 55 Lymph Node 5

+ 7.0 + + 8.6 + 6.0 + + 8.5 - 4 . 0 - 4.0

+ 5.0 + + cont. a + 7.0 + + 8.0 - 4 . 0 - 4.0

+ 5.0 + + 8.5 - 4 - 4

+ 6.0 + + 7.0 - 4 . 0 - 4.0

-- 4 + + cont. d - - 6 . 0 + + 8.7

+ + 7.0 + + 8.0 - cont. d -- cont. a

aEstablished by viable counts. ~' + , weak colour reaction (0.2 < A414 • 0.8 ) ; "~- ~-, strong colour reaction (A4~4 > 0.8); --, no reac- tion (A414 < 0.2). "Log~o c.f.u, ml- i. dOvergrowth by contaminants.

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that were positive. C-ELISA results were available 2-3 days before colonies appeared on culture plates.

DISCUSSION

Our results demonstrate that the C-ELISA can be a sensitive method for the rapid detection of Brucella in enrichment liquid cultures of blood or tissues. C- ELISA and cultural isolation results correlated (100%) in our small number of simulated samples. Combined enrichment-C-ELISA may provide more rapid diagnosis, especially for blood cultures.

Hydrophobic cloths such as polyester have advantages over non-porous sup- ports for solid-phase ELISA tests. As compared to non-porous solid phases such as polystyrene plates, the macroporous hydrophobic cloths absorb larger sample volumes, providing a large surface for immunoreactions (increasing reaction rates and potentially, sensitivity) and are easily washed without spe- cial apparatus. These characteristics make the C-ELISA an ideal test for an- tigens such as pathogens in enrichment cultures. Such enrichment procedures are used in the detection of Salmonella in food samples by conventional ELISA (Ibrahim et al., 1986).

Bovine anti-Brucella serum contains other hydrophobic proteins in addition to anti-Brucella Ig. Heating antiserum 36398N-2 at 75 ° C for 10 min may have increased the adsorption of Ig onto polyester cloth, as shown by the enhanced signal obtained in the C-ELISA (Fig. 1 ). It has been shown that the Fc region of the IgG molecule is much more susceptible to denaturation by heat than the Fab region (Conradie et al., 1983 ). Partial heat denaturation may increase IgG adsorption onto the hydrophobic cloth surface by exposing hydrophobic sites of the Fc. The use of diluted whole bovine antiserum as a source of capturing antibody makes the C-ELISA a simple and economical method. Antibody- coated cloths and H R P C were stable at 4 °C for several months.

Small numbers of Brucella were unable to initiate growth in enrichment cul- tures of eight of the 14 homogenized bovine tissues tested and this was the cause of' failure to detect the Brucella by the enr ichment-C-ELISA test. This may be due to the presence of inhibitory substances in such tissues, the effects of' which might be removed by diluting these suspensions before culture. Ad- dition of selective antibiotics to suppress competing contaminating flora would be useful in improving isolation by enrichment culture and this should be stud- ied as the next step in the development of the rapid C-ELISA for BruceUa. The C-ELISA was positive for all cultures which yielded brucellae. No false posi- tives occurred in 20 negative samples, even though six of these were heavily contaminated with other bacteria. Potential false-positive reactions occurring with Yersinia enterocolitica (serotype 09) might be eliminated by the use of a monoclonal antibody to Brucella antigen instead of the polyclonal reagent an- tibodies used presently. In naturally infected tissue specimens, most BruceUa

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organisms are likely to be located intracellularly and it may, therefore, be nec- essary to more fully disrupt tissue samples prior to enrichment.

The sensitivity of the C-ELISA could be further improved by amplification of the enzyme response, and increasing the purity of the antibody and the molar ratio of enzyme to antibody in the conjugate. Work is currently under way to evaluate and improve the sensitivity of the C-ELISA in the diagnosis of brucellosis.

ACKNOWLEDGEMENTS

We thank Drs. K. Nielsen and P.F. Wright for helpful advice and the late N. Rab for providing B. abortus 413 cells. This work was partially supported by a Natural Sciences and Engineering Research Council of Canada Operating Grant (A4698) to H.Y.

REFERENCES

Batra, H., Chand, P., ThillaiKoothan, P. and Talwar, G., 1987. Coaggulation test with coloured Staphylococcus aureus for detection of brucella antigens in cattle brucellosis. Vet. Rec., 121: 65-66.

Chand, P., Gupta, R., Khanna, R. and Sudana, J., 1987. Detection of brucella antigen in bovine fetal stomach contents by agar gel precipitation and counter-immunoelectrophoresis. Res. Vet. Sci., 43: 132-133.

Conradie, J.P., Grovender, M. and Visser, L., 1983. ELISA solid phase: Partial denaturation of coating antibody yields a more efficient solid phase. J. Immunol. Methods, 59: 289.

Ibrahim, G., Lyons, M., Walker, R. and Fleet, G., 1986. Rapid detection of salmonellae in foods using immunoassay systems. J. Food Prod. 49: 91.

Lowry, 0., Rosenbrough, N., Farr, A. and Randall, R., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265.

Meador, V., Tabatabai, L., Hagemoser, W. and Deyoe, B., 1986. Identification ofBrucel la abortus in formalin-fixed, paraffin-embedded tissues of cows, goats, and mice with an avidin-biotin- peroxidase complex immunoenzymatic staining technique. Am. J. Vet Res., 47:2147-2150.

Nakane, P.K. and Kawaoi, A., 1974. Peroxidase-labeled antibody: a new method of conjugation. J. Histochem. Cytochem., 22: 1084.

Perera, V., Creasy, M. and Winter, A., 1983. Nylon bead ELISA for the detection of sub-picogram quantities of Brucella antigens. J. Clin. Microbiol., 18: 601.

Read, R.S.D., Cox, J.C., Ward, H.A. and Nairn, R.C., 1974. Conditions for purification of anti- Brucella antibodies by immunoadsorption and elution. Immunochemistry, 11: 819-822.

Yolken, R., 1982. Enzyme immunoassays for the detection of antigens in body fluids: current limitations and future prospects. Rev. Infect. Dis., 4: 35.