Enzyme immunoassay for the detection of antibody to Pseudomonas pseudomallei exotoxin in mice
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FEMS ,Microbiology Letters 40 (1987) 27-31 27 Published by Elsevier
Enzyme immunoassay for the detection of antibody to Pseudomonas pseudomallei exotoxin in mice
G. Ismai l a, M. Noor Embi a, O. Omar a, N. Razak a J.C. Al len and C.J. Smith
Research Division, North East Wales Institute, Deeside, Clw~,d, U.K., and a The National University of Malaysia, Sabah Campus. Kota Kinabalu, Malaysia
Received 4 July 1986 Accepted 20 August 1986
Key words: Pseudomonas pseudomallei; Exotoxin; Antibody; (Detection; Enzyme immunoassay)
An enzyme immunoassay (enzyme-linked im- munosorbent assay; ELISA) to detect mouse anti- body to exotoxin of Pseudomonas pseudomallei was developed in which exotoxin preparations were used to coat the solid phase. The specificity of the assay was supported by inhibition assays with the toxin preparations. Sera from immunised mice and supernatants from antitoxin-producing hy- bridoma cells were tested by this technique, and the ELISA described appears to provide a sensi- tive, specific, and practical method for the de- termination of (a) antibody to exotoxin, and (b) concentration of exotoxin. Both systems may prove invaluable in the diagnosis of melioidosis and in epidemiological studies of melioidosis in endemic areas.
ease of man and animals in South east Asia and tropical Australia. The role of toxic products of P. pseudomallei in the pathogenesis of melioidosis has been the focus of interest of many investiga- tors in the past [1-4]. These studies indicated that cell-free filtrates of the organism possess varied biological activities which include lethal toxicity and necrotising, proteolytic and lecithinase activi- ties. However, it is still unclear whether the dis- ease melioidosis is toxin-mediated. We have now isolated the exotoxin of P. pseudomallei, which accounts for the lethal toxicity in mice, in a rea- sonably pure form. The objective of this study was to prepare a specific monoclonal antitoxin for use in an enzyme-linked immunosorbent assay which itself would be of use for the detection and de- termination of the exotoxin.
P. pseudomallei is the causative agent of melioidosis, a fulminating and usually lethal dis-
Correspondence to: C.J. Smith, Research Division, North Fast Wales Institute, Deeside, Clwyd, CH5 4BR, U.K.
3. MATERIALS AND METHODS
3.1. Bacteria P. pseudomallei used in this study was obtained
from the Microbiology Department, Faculty of Medicine, National University of Malaysia, Kuala Lumpur. This strain was isolated from a male patient who died of P. pseudomallei infection. The
0378-1097/87/$03.50 1987 Federation of European ,Microbiological Societies
organisms were routinely propagated on Brain Heart Infusion agar (BHIA) slants and stored at 4C.
3.2. Isolation of P. pseudomallei exotoxin P. pseudomallei was grown in Brain Heart Infu-
sion broth (BHIB) containing 1% (w/v) mucin and 2% (v/v) glycerol at 37C under static condi- tions for 7 days. The culture was harvested by centrifugation at 32000 x g for 40 rain (Sorvall RL-53 superspeed, 14000 rev./min, Rotor GSA). The resulting supernatant was centrifuged at 55000 x g for 40 min (Beckman L 3-50 Ultra- centrifuge, 30000 rev./min, Rotor 50.2 Ti). The supernatant was then millipore-filtered (0.45 #m) to remove any remaining cells. The cell-free filtrate was dialysed twice against phosphate-buffered saline (PBS) pH 7.4, for 24 h. The resulting crude toxin extract was concentrated to a volume of 20-30 ml through an ultra-filtration unit (Cole Palmer) with a cut-off point of 10 kDa using a nitrogen pressure of not more than 20 p.s.i. At the end of the concentration step, the sample was lyophilised and the powder was stored at -20C until further use. In the final step of exotoxin isolation, the freeze-dried sample was resuspended in PBS and applied to a G100 Sephadex column. The single peak showing toxic activity in the mouse lethality test was collected and stored at -20C.
Toxicity of the exotoxin preparation was de- termined at each stage of the purification by in- traperitoneal injection of 0.5 ml of serially diluted test samples into groups of mice. Mortality was recorded for 7 days and the LDs0 was calculated by the method of Reed and Muench .
3.3. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS- PA GE)
Samples of exotoxin preparations (50 ttl, 1-5 mg/ml) were assayed for purity by SDS-PAGE (12.5%) as described previously . The electro- phoresis was stopped when the tracking dye (bromophenol blue) reached the end of the gel. The gel was stained with Coomassie brilliant blue for 24 h and destained for several days according to the method described by Chrambach et al. . Samples were assayed for protein content prior to electrophoresis. Protein content was 250 #g/mg.
3. 4. A ntiserum production Mice were immunised with a solution of 250 ~g
protein/ml exotoxin preparation mixed with an equal volume of Freund's complete adjuvant. The immunization schedule used was as described by Galfre and Milstein . Mice were test-bled routinely, and those producing high titres were selected for fusion. Sera from exsanguinated mice were collected and used as positive controls in subsequent assays.
Hybridoma production was carried out using a modification of the polyethylene glycol technique described by Kohler and Milstein . The modifi- cations employed were based on those outlined by Fazekas de St. Groth and Scheidegger . Anti- body-producing hybrids were selected by cloning in soft agar and the clones expanded in vitro. Spent culture medium containing positive anti- body activity was used in all assays.
3.5. ELISA method The ELISA was performed according to a mod-
ification of the method of Voller et al. . Poly- styrene flat-bottomed 96-well microtitre plates were used for the assay (Nunc lmmunoplate II, Gibco Laboratories, Grand Island, NY, U.S.A.). Wells were coated with 100 ~1 of purified exotoxin (0.5 ffg/ml in carbonate/bicarbonate binding buffer, 0.1 M, pH 9.6). After an overnight incuba- tion at 4C, the plates were washed 5 times with PBS containing 0.05% Tween 80 (PBST) and stored at 4C until used. A 100-/~1 sample of antiserum diluted in PBS was added to the appropriate wells and incubated, shaking, at room temperature for 30 min. After incubation, the plates were washed 5 times with PBST, and 100 ~tl of peroxidaselabelled rabbit anti-mouse immunoglobulin G (Sigma, St. Louis, U.S.A.), diluted 1 : 200 in PBST was added.
The plates were incubated for 20 min with shaking, at room temperature, washed 5 times with PBST, and 100 ~1 of substrate solution was added. The substrate solution used was 0.012% H202 in citrate buffer (pH 5.0) containing 6 mg of 2-2'-azino-bis (3-ethylbenzthiazoline sulfonic acid) (ABTS) per ml. Reaction mixtures were incubated with shaking at room temperature for 10 min. Control wells were treated identically, except that PBS replaced the serum. Absorbance at 405 nm (A405) was read in a Microelisa Reader (Dy- natech).
A competitive ELISA test was performed by the method of Yoiken and Stopa . A dilution of the test serum in PBS was selected which gave an absorbance of 1.00-1.20 in wells coated with exotoxin. The serum was pre-incubated with in- creasing concentrations of exotoxin for 30 min at 37C, and then reacted in the ELISA system. The reduction in absorbance with the addition of ex- otoxin was expressed as a percentage of the A reading for a control containing no exotoxin. Re- actions showing an inhibition of 30% or more of the controls were considered to be positive.
4.1. Purification of exotoxin Fig. 1 illustrates the elution profile of partially
purified exotoxin subjected to Sephadex G100 gel permeation chromatography.
One major peak was observed which eluted at fractions 22-26. This peak was identified as the exotoxin fraction by the mouse lethality test. The LDs0 of the preparation varied between 20-30 #g protein, depending on the batch. The 2 smaller peaks eluting at and just after the void volume of the column contained no toxin, as judged by the mouse lethality test. The lypholised exotoxin pool could be stored at 4C, and each preparation retained full lethal toxicity for more than 6 months.
To evaluate the degree of purity of the ex-
~ 0. ,
8 12 16 20 24 28 32 3"6
FRACTIONS / 5ml
Fig. t. Sephadcx G-100 chromatography of the exotoxJn pool Fractions of about 5.0 m| were collected at a flow rate of 5 m]/h. Protein was measured at 280 nm.
- " *45
- I PD , -31
Fig. 2. SDS-PAGE analysis of purified exotoxin (Lane A) and M~ ( 10-3) markers (Lane B). Arrow indicates 31-kDa pro- tein in the exotoxin preparation.
otoxin, the purified preparation containing ap- prox. 50 ~g of protein was subjected to SDS-PAGE. The Coomassie-blue-stained gel showed only one major band at approx. 31 kDa (Fig. 2) with negligible amounts of contaminating proteins.
4.2. Titration of mouse sera and hybridoma super- natant (CB4)
Titration studies using exotoxin-immunised mouse serum and hybridoma supernatant revealed a linear relationship between sera or supernatant dilution and net absorbance (Fig. 3). The mouse immune sera and the CB4 supernatant from the hybridoma culture gave a high titre with P. pseu- domallei exotoxin in a direct ELISA for antibody to toxin. Indeed, in both cases, a significant posi- tive ELISA reaction was observed, even at dilu- tions of 1.6 104, when the minimal usable ab- sorbance value was fixed at a reading equivalent
O9 0.6 O~
,~ x 0
~.4 \ \
0 .2 \% 0
I I 1 I I I I 20C0 8C:)0 57000 i 2SOOt
Fig. 3. Relationship between absorbance reading (A,~) 5) and serial 2-fold dilutions of mouse immune scrum (e) and anti- toxin hybridoma supernatant (O1.
to twice that obtained from negative control as- says.
The reactivity of mouse sera and antibody-con- taining hybridoma supernatants was unaffected by pre-adsorption with bacterial growth medium comprising BHIB, 1% mucin and 2% glycerol (data not shown).
The titration results indicated that the optimum antibody dilution for use in an inhibition assay for exotoxin would be 1:2000. Subsequently, this di- lution was used in our competitive ELISA studies
IO0 0.016 0.08 0.4 2.0 IO 50 I00
COMPET ITOR XOTOXIN ( .ug/ml)
Fig. 4. Quantitative inhibition of ELISA reactivity with puri- fied exotoxin using the reference CB4 hybridoma supernatant at a dilution of I :2000.
and a positive control at this dilution was included in every test plate, thus giving a standard refer- ence value for all the results.
Fig. 4 shows the results of a typical competitive ELISA in which P. pseudomallei exotoxin was used as the competitor antigen. The binding of the positive control supernatant was inhibited 94% by purified exotoxin at a concentration of 50 #g/ml. The detection limits of the assay therefore ranged from 0.016-50/lg/ml.
An important limitation in the evaluation and diagnosis of human melioidosis has been the ab- sence of suitable serological assay procedures. Pre- viously reported serological assays for the detec- tion of P. pseudornallei infection have not been widely used for a variety of reasons. One of the more frequently used tests is the haemagglutina- tion assay, which utilises crude antigen extracts from sonicated suspensions of ~P. pseudomallei. Although this haemagglutination test has proved to be highly sensitive and has been used for the diagnosis of melioidosis, it lacks specificity in that positive reactions with P. aeruginosa have been observed . The complement-fixation test has also been used to detect possible subclinical melioidosis in population surveys . However, the test lacks sensitivity and is thus incapable of detecting low levels of specific antibody to P. pseudornallei.
ELISA. introduced by Engvall and Perlmann for the quantitative determination of antibodies  has been applied to the diagnosis of bacterial [16-18], viral [19,20] and parasitic  diseases. The use of ELISA to measure antibodies specific for P, pseudomallei antigen has not been previ- ously reported.
In order to develop this assay, it was necessary to prepare and characterise essentially pure ex- otoxin for use as an immunogen. We describe here a simple method that has resulted in the isolation of a reasonably pure P. pseudomallei exotoxin, shown to be both pure and active. Analysis by SDS-PAGE indicated that the preparation con- sisted of a single major protein which, when in-
jected into mice, elicited the production of a specific antitoxin antibody. Using this purified exotoxin, we have raised a monoclonal antibody in sufficient amounts to further develop a highly sensitive and specific ELISA technique.
Possible applications of the ELISA methods described here, some of which are being studied, are numerous. The most immediate will be in studies attempting to delineate the role of ex- otoxin in P. pseudomallei infection. For example, detection of antibody specifically directed at P. pseudomallei exotoxin in serum of melioidosis pa- tients can indirectly provide evidence for in vivo exotoxin production during infection. If indeed exotoxin does play an important part in the path- ogenesis of melioidosis, detection of antitoxin an- tibody can be potentially useful for seroepidemio- logic surveys of human melioidosis; especially in procuring evidence of subclinical or undiagnosed infections in endemic areas.
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