Vol. 57, No. 3
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1991, p. 836-842
0099-2240/91/030836-07$02.00/0 Copyright © 1991, American Society for Microbiology
Rapid and Sensitive Sandwich Enzyme-Linked Immunosorbent Assay for Detection of Staphylococcal Enterotoxin B in Cheese CELINE MORISSETTE,1 2t* JACQUES GOULET,2 AND GILLES LAMOUREUX' Centre de Recherche en Immunologie, Institut Armand-Frappier, Laval, Quebec H7V 1B7,1 and Departement de Sciences et
Technologie des Aliments, Pavillon Paul-Comtois, Universite Laval, Sainte-Foy, Quebec GIK 7P4, Canada Received 25 June 1990/Accepted 10 December 1990
A rapid and sensitive screening sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of staphylococcal enterotoxin B (SEB) in cheese by using a highly avid anti-SEB antibody (Ab) as the capture Ab (CAb) and as the biotinylated Ab conjugate. The glutaraldehyde fixation method for the immobilization of CAb on polystyrene dipsticks was superior to the adsorption fixation and the adsorptionglutaraldehyde fixation methods. The glutaraldehyde fixation method resulted in a higher surface-saturating CAb concentration as evaluated by the peroxidase saturation technique and by the ability of the CAb-coated dipstick to discriminate between positive and negative controls (index of discrimination). Of nine blocking agents used alone or in pairs, lysine-human serum albumin, bovine serum albumin, human serum albumin, and gelatin effectively saturated available sites on the CAb-coated dipsticks without causing interference with the antigen-Ab reactions. The addition of 1% polyethylene glycol to the diluent of the biotinylated anti-SEB Ab conjugate improved the detection of SEB. A concentration of 4% polyethylene glycol allowed a 5-min reaction time for the streptavidin-biotin-horseradish peroxidase conjugate. Cheddar cheese homogenate reduced the sensitivity of the SEB assay; however, the sensitivity was restored when 1.6% (wt/vol) of either a nonionic detergent (Mega-9) or two zwitterionic detergents (Zwittergent 3-10 and 3-12 detergent) was added to the diluent. By using the rapid sandwich ELISA, a minimum of 0.5 to 1.0 ng of SEB per ml was detected within 45 min. The whole procedure for the analysis of the cheddar cheese samples was completed within 1 h. This sandwich ELISA could be a rapid and sensitive screening method for the detection of staphylococcal enterotoxins in food for the agri-food industries.
Staphylococcal enterotoxins (SE) are one of the leading causes of bacterial food poisoning in Canada (36). These toxins in the active state are resistant to both proteolytic enzymes, such as trypsin, chymotrypsin, rennin, and papain (2), and high temperatures (boiling crude solutions for 30 min) (3). They have been found in organic raw materials as well as pasteurized, cooked, or otherwise processed foods (25). Rapid screening of SE for quality control in the food industry relies on rapid and sensitive methods. Many enzyme immunoassay techniques have been described for the detection of SE in culture supernatants and in food samples (1, 9-11, 13, 18, 24, 26, 30, 32, 37-39). The enzyme-linked immunosorbent assay (ELISA) is as efficient and sensitive as the radioimmunoassay but does not require the use of radioactive materials. Four types of ELISA were studied for their ability to detect SE in food samples (9). Whereas the competitive ELISA showed a higher specificity and was not sensitive to protein A in food extracts, the sandwich ELISA with labeled antibody (Ab) gave the best overall performance. Sandwich ELISAs are commonly performed by immobilizing a capture Ab (CAb) on plastic supports. The antigen (Ag) captured by the CAb support may be detected either by an enzyme-labeled Ab specific for the same determinant as the CAb or by an enzyme-labeled Ab recognizing a different epitope on the captured, multivalent Ag (4, 5). Effective pairs of monoclonal Abs and polyclonal Abs can be used in the sandwich ELISA to detect SE (20, 34). The performance
of the ELISA depends on the apparent affinity (avidity) and specificity of the selected Ab. This article describes the development of a sandwich ELISA to be used as a rapid screening assay for the detection of SE in food. By using avid polyclonal Abs to staphylococcal enterotoxin B (SEB) and a biotin-streptavidin amplification system, each major step of the analytical procedure (immobilization method of CAb, diluent for reagents, incubation period, and diluent for food preparation) was optimized and standardized to improve the sensitivity and the reaction rate of the test. MATERIALS AND METHODS SEB and Ab to SEB. SEB (Sigma Chemical Co., St. Louis, Mo.; lots 32F-4024 and 94F-4001) was used as the Ag. The preparation was rehydrated in sterile distilled water at a concentration of 1.5 mg of SEB per ml. Rabbit anti-SEB Ab containing the crude immunoglobulin fraction of antiserum was purchased from Sigma (lot 91F-40171). Diluents. Phosphate-buffered saline (0.01 M sodium phosphate-0.15 M NaCl, pH 7.4) (PBS) containing 0.02% sodium azide (NaN3) and supplemented with 0.05% Tween 20, 0.1% bovine serum albumin (BSA), and 4% polyethylene glycol 6000 (PEG) was used as a general diluent. In some experiments, the concentration of PEG was varied. Physiological saline containing 0.9% NaCl, 0.05% Tween 20, and 0.01% NaN3 was used as a washing solution. Biotinylated Abs. The anti-SEB Ab was biotinylated by use of the procedure of GIBCO-BRL (Burlington, Ontario, Canada). Biotin-N-hydroxysuccinimide ester (Bethesda Research Laboratories/Life Technologies, Inc., Gaithersburg, Md.; lot 51101) was dissolved to a concentration of 50 mg/ml
* Corresponding author. t Present address: Food Research Centre, Research Branch, Agriculture Canada, Central Experimental Farm, Ottawa, Ontario
KlA 0C6, Canada.
836
VOL . 57 i
RAPID DETECTION OF SEB IN CHEESE
1991
TABLE 1. Molecular weight, critical micellar concentration, and ionic state of detergents testeda Mol wt
Critical micellar concn (M)
Hexyl-p-D-glucopyranoside
264.3
Large
Nonyl-p-D-glucopyranoside
306.4 510.6
Detergent
Ionic state
Detergent test kit
Heptyl-3-D-glucopyranoside 278.3 Octyl-f3-D-glucopyranoside 292.4 Dodecyl-,B-D-maltoside Mega test kit Mega-8 Mega-9
321.5 335.5
Zwittergent test kit Zwittergent 3-08 detergent Zwittergent 3-10 detergent Zwittergent 3-12 detergent Zwittergent 3-14 detergent
279.6 307.6 335.6 363.6
Other common detergents Sodium dodecyl sulfate Tween 20 Dishwashing liquid (Ivory)
288.4
2.5 x 10-2 6.5 x 10-3
Nonionic Nonionic Nonionic Nonionic Nonionic Nonionic Nonionic
Large 3.9 x 10-2 3.6 x 10-3 3.3 x 10-4
Zwitterionic Zwitterionic Zwitterionic
5.9 x 10-5
Ionic Nonionic
Zwitterionic
a From Calbiochem Biochemical & Immunochemical catalog 87, Helenius et al. (14), and Gardas and Lewartowska (12).
in dry N,N-dimethyl formamide (Fisher Scientific Co., Orangeburg, N.Y.; catalog no. D-119). A 10-pul volume of this solution was reacted with 0.125 ml of protein (20 mg/ml), 20 p.l of 0.5 M NaHCO3 (pH 9.0), and 40 p.l of H20 for 1 h at room temperature with gentle agitation. The reaction was stopped by adding 25 p. of 1 M NH4Cl. The mixture was dialyzed against PBS and membrane filtered (pore size, 0.45
Am).
A biotinylated donkey anti-rabbit immunoglobulin (Amersham Canada Ltd., Oakville, Ontario, Canada; catalog no. RPN 1004, lot 30) was also used to detect the presence of rabbit Abs on dipsticks. Streptavidin-biotinylated enzyme complex. Biotinylated anti-rabbit immunoglobulin and biotinylated anti-SEB Ab were detected by a preformed streptavidin-biotinylated horseradish peroxidase complex from Amersham Canada (catalog no. RPN 1051, lot 42 and 61). Enzyme substrate. A horseradish peroxidase substrate solution was freshly prepared by dissolving 100 mg of o-diaminobenzene dihydrochloride (Sigma) in 100 ml of 0.2 M Na2-phosphate-0.1 M citrate buffer, pH 5, containing 150 p.l of 30% H202. The peroxidase reaction was stopped by the addition of 0.5 ml of a solution containing 2 M H2SO4 and 0.1 M NaSO3 (27). Detergents. Several detergents (Table 1) were used as substitutes for Tween 20 in the diluent for the preparation of food homogenates. All selected detergents were soluble in PBS-NaN3-BSA-4% PEG. The Detergent Test Kit, Mega Test Kit, and Zwittergent Test Kit were obtained from Calbiochem Biochemical & Immunochemical (Terochem Laboratories Ltd., Edmonton, Alberta, Canada). Sodium dodecyl sulfate was obtained from Matheson Coleman & Bell, Norwood, Ohio, and Tween 20, polyoxyethylene sorbitan monolaureate, was obtained from Fisher Scientific. Immobilization of CAbs on polystyrene dipsticks. (i) Adsorption fixation method. A batch of 500 polystyrene dipsticks (Sarstedt no. 81,970, Ruhr, West Germany) was washed with deionized water for 2 h and incubated overnight
837
in a 0.01 M carbonate buffer (pH 9.6) containing increasing concentrations (0, 0.5, 1, 5, 10, and 25 p.g/ml) of CAb. In some experiments, the remaining active sites on the CAbcoated dipsticks were saturated for 2 h with a solution of blocking agent (1% BSA in PBS). All incubations were performed at room temperature (22°C) with agitation on a rotary shaker. After two washings with PBS-NaN3-Tween 20, the CAb-coated dipsticks were stored in PBS-NaN3Tween 20 at 4°C until required. (ii) Combined adsorption-glutaraldehyde fixation method. The adsorption-glutaraldehyde fixation method was adapted from the binding technique of Saunders and Clinard (31). A batch of 500 polystyrene dipsticks previously washed with deionized water for 2 h was incubated for 1 h in a 0.01 M carbonate buffer (pH 9.6) containing increasing concentrations (0, 0.5, 1, 5, 10, and 25 pug/ml) of CAb. The CAb-coated dipsticks were then incubated with 0.25% (vol/vol) glutaraldehyde in PBS (pH 7.0) for 30 min, washed four times with distilled water, and reincubated overnight in the same CAbcontaining solutions. In some experiments, the remaining active sites of the CAb-coated dipsticks were saturated with a solution of blocking agent (1% BSA in PBS) for 2 h. All incubations were performed at room temperature (22°C) with agitation on a rotary shaker. After two washings with PBS-NaN3-Tween 20, the CAb-coated dipsticks were stored in PBS-NaN3-Tween 20 at 4°C until required. (iii) Glutaraldehyde fixation method. The glutaraldehyde fixation method was adapted from the method of Suter (33) by the method of Tijssen (35). A batch of 500 polystyrene dipsticks was washed twice with distilled water and once with 0.1 M sodium phosphate buffer (pH 5). The dipsticks were pretreated with 0.2% glutaraldehyde (Sigma; catalog no. G-6257, lot 116F-5027, grade II) in 0.1 M sodium phosphate buffer (pH 5) for 4 h. The dipsticks were washed three times with the same buffer and incubated in 0.1 M sodium phosphate buffer (pH 8) containing increasing concentrations (0, 2, 4, 8, 10, 20, 40, and 60 p.g/ml) of CAb for 14 to 16 h. The CAb-coated dipsticks were then washed three times with PBS (pH 7.4), and in some experiments the non-Ab-occupied sites were saturated by incubating dipsticks for 2 h in a solution of blocking agent (100 mM lysine) in PBS. All incubations were performed at room temperature (22°C) with agitation on a rotary shaker. After two washings with PBS (pH 7.4), the dipsticks were stored in PBS-NaN3Tween 20 at 4°C until required. Rapid sandwich ELISA. In a typical assay (Fig. 1), a CAb-coated dipstick was incubated with 1.0 ml of SEcontaminated sample for 5 min. All incubation steps were performed at room temperature with agitation. After two washings with the washing solution, the CAb-coated dipstick was transferred to a tube containing 300 p.1 of a dilution of biotinylated anti-SE Ab conjugates. After 5 min of incubation, the dipstick was washed twice and transferred to a tube containing 300 pul of diluted streptavidin-biotin-horseradish peroxidase complex. The CAb-coated dipstick was removed after 5 min, washed twice, and transferred to a tube containing 1.0 ml of H202-o-diaminobenzene dihydrochloride for 5 min. After removing the dipstick, the reaction was stopped by transferring the reaction mixture into tubes containing the stopping solution. The optical density of the solution at 490 nm (OD49) was read with an optical fiber probe (PC 800; Brinkmann [Canada Ltd.], Rexdale, Ontario, Canada). Blocking treatment. Several reagents (Table 2) were tested for their ability to block nonspecific binding and saturate the unoccupied sites on surface-immobilized CAb. They were used alone or in pairs at a concentration of 1% in PBS. After
,.
_A
APPL. ENVIRON. MICROBIOL.
MORISSETTE ET AL.
838
Step
1
s. .
C'0 0
.
0
Step 2
Step 3
*
A
A
TABLE 2. Reagents used as blocking agents after immobilization of Abs on polystyrene dipsticks
Sigma (catalog no. L-6001) Fisher (catalog no. G-46) albumin .........I n-house preparation (Institut
DL-Lysine ................ Glycine ................. Human serum 0
Io
Armand-Frappier) Miles Laboratories, Inc., Naperville, Ill. (catalog no. 81-066-1) Whole rabbit serum .............I n-house preparation (single bleed from one animal) Bacto-Peptone ................ Difco (catalog no. 0118-05-4) Casein ................ Sigma (catalog no. C-5890) Gelatin ................ Sigma; from calf skin type IV (catalog no. G-0510) Organosilane (Prosil-28) ....... SCM Chemicals, Gainesville, Fla. (catalog no. 11975-0) BSA .................
,S 20 minutes
10 minutes
Washing
Washing
-4
Washing
CAb-coated dipsticks was employed to discriminate between positive and negative controls. Peroxidase saturation technique. The peroxidase saturation technique of Munioz et al. (22) was used to estimate the Ab-free surface sites after the immobilization. Horseradish peroxidase (Boehringer GmbH; lot 10300224-40, 308 U/mg) diluted to 0.1 mg/ml in PBS was incubated with CAb-coated dipsticks for 2 h at room temperature with agitation. The dipsticks were then washed four times with PBS, and the enzyme activity was developed as described above. Preparation of cheese samples. Five-gram samples of cheddar cheese contaminated with 200 ng of SEB per g were homogenized in a blender for 15 s with 10 ml of diluent containing various concentrations (0.016, 0.16, and 1.6% [wt/vol]) of detergents (Table 1). A 1.0-ml aliquot of the homogenate was used for testing with the rapid sandwich ELISA. Index of discrimination. To evaluate the immunoreactivity of the CAb-coated dipsticks in the rapid sandwich ELISA, we used a discrimination index defined as follows: (OD490 of
positive control - OD490 of negative control)/OD490 of negative control x 100 = %.
_
t ': ..;
.-. A:..
:.-.
. o
OD 490
SE Antibody against SE Biotinylated anti-SE Ab conjugate A _ s_%b
Source
Reagent
_
_
t
._s2_
FIG. 1. Major steps of the rapid sandwich ELISA procedure. OPD, o-Diaminobenzene dihydrochloride.
immobilization by using an unsaturating concentration of CAb (4 ,ug/ml), the dipsticks were incubated with the blocking agents for 2 h and washed three times. The capacity of the blocking agents to saturate the Ab-free sites on CAbcoated dipsticks was evaluated by the peroxidase saturation technique. The rapid sandwich ELISA with the blocked
RESULTS Selection of Abs. The anti-SEB Ab used in this study was selected for its high avidity for SEB. The anti-SEB Ab ELISA titer of the crude immunoglobulin fraction was 409,600 at the endpoint of negative control, when incubated for a 5-min period with SEB-coated dipsticks and a dilution of the protein A-horseradish peroxidase conjugate (21). Selection of a method to immobilize the CAb. Three immobilization methods, adsorption fixation, adsorption-glutaraldehyde fixation, and glutaraldehyde fixation, were compared for their ability to bind CAb to polystyrene dipsticks under saturating conditions and for the efficiency of the resulting CAb-coated dipsticks to discriminate between negative and positive controls. After the immobilization of CAb, residual free sites were evaluated by the peroxidase saturation technique. In this technique, the OD value is directly proportional to the available binding sites on support. Accordingly, the OD values decreased as the CAb concentration increased, and regression lines for the rapidly decreasing OD readings and for the slowly decreasing (near-constant) OD readings were calculated (Fig. 2). By using the glutaraldehyde fixation method, the surface-saturating CAb concentration was evaluated as the intersection point between both regression lines (22), i.e., 11 ,ug/ml. The adsorption fixation
RAPID DETECTION OF SEB IN CHEESE
VOL. 57, 1991
0.7
900 ,2 800 O 700 > 600 2 500 a: ) 400 D 300 0 200
E
0.6 z 0.5 z < 0.4 0 0.4 00.3 0.3
0
.. .. .. .. 60 20 30 40 50 10 CONCENTRATION OF CAb (ligtmQ
FIG. 2. Immobilization of increasing concentrations of CAb with the glutaraldehyde fixation method. Non-Ab-occupied surface sites were evaluated by the peroxidase saturation technique (n = 8, arithmetic mean of OD values from a test repeated four times in duplicate).
839
GA-CAb
GA-CAb-Lysirw
v
0 100
z
0
0
10
20
30
40
50
60
CONCENTRATION OF CAb (igml) FIG. 4. Immunoreactivity of CAb-coated dipsticks as estimated by the index of discrimination. GA, Glutaraldehyde fixation method.
and adsorption-glutaraldehyde fixation methods demonstrated a surface-saturating CAb concentration of approximately 5 ,ug/ml, a twofold decrease compared with the concentration demonstrated by the glutaraldehyde fixation method. The ability to discriminate between positive and negative controls with the CAb-coated dipsticks obtained from the glutaraldehyde fixation method is represented in Fig. 3 and 4. A maximum immunoreactivity was observed at about 10 jig of CAb per ml. Results expressed as the index of discrimination allow visualization of the surface-saturating CAb concentration where the OD value of the positive control is increased while the OD value of the negative control remains low. The index of discrimination for the adsorption fixation and the adsorption-glutaraldehyde fixation methods was at best 94 and 265%, respectively, at a concentration of 5 ,ug of CAb per ml. The adsorptionglutaraldehyde fixation and the glutaraldehyde fixation methods gave good discrimination at their surface-saturating CAb concentration; however, the glutaraldehyde fixation method was preferred because of its higher index of discrimination and its higher surface-saturating CAb concentration. How1.2
Negative control GA-CAb &
E
---
GA-CAb-Lysne
010
Positive control
*
F _iir ~GACAb_
-
< 0.8
GA-CAb-Lysin
0.6 w ..j
0.4
0 0
30 40 50 20 CONCENTRATION OF CAb (1tgIml)
10
60
FIG. 3. Immunoreactivity of CAb-coated dipsticks evaluated by the detection of a positive control (laboratory diluent containing 145 ng of SEB per ml) and a negative control (laboratory diluent) by rapid sandwich ELISA (arithmetic mean of OD values from a test performed in duplicate). GA, Glutaraldehyde fixation method.
ever, when this latter method was used with the suggested blocking agent, lysine (35), interference was observed on the detection of SEB (Fig. 3 and 4). Selection of a blocking agent. Preliminary tests were conducted with a series of nine blocking agents used alone or in pairs and with dipsticks coated with 4 jig of anti-SEB Ab per ml (undersaturating CAb concentration). Table 3 shows the capacity of the four best blocking agents to inhibit the binding of the peroxidase and their influence over the immunoreactivity of the CAb-coated dipsticks in the rapid sandwich ELISA. The index of discrimination of the CAbcoated dipsticks blocked with those agents was similar to the one obtained with the unblocked controls, indicating that their effect is not detrimental to the immunological reaction in undersaturating conditions. The following other blocking agents gave either a high OD reading with the peroxidase saturation technique (do not saturate available sites) or poor index of discrimination (interfere with the further reactions): lysine, lysine-organosilane, lysine-BSA, lysine-glycine, glycine, glycine-organosilane, glycine-human serum albumin, peptone, peptone-lysine, peptone-glycine, and whole rabbit serum. At the optimal coating CAb concentration (11 ,ug of anti-SEB Ab per ml), the effect of the four best blocking agents was maintained. Because of its high index of discrimination and its low OD value for negative control, gelatin was selected as the best blocking agent for high dilutions of biotinylated anti-SEB Ab. Selection of the incubation periods. Minimal incubation periods were selected to detect the critical enterotoxin concentration (100 ng of SE per 100 g of food or 0.5 ng of SE per ml of food homogenate) assuming a dilution factor of 2. This minimum concentration was detected when the period of incubation of CAb-coated dipstick with the SEB-containing sample was longer than 10 min; for an incubation time of 20 min, the minimal detection limit is around 0.5 ng of SEB per ml (Table 4). This period of incubation can be increased when the dilution factor is higher than 2. Effect of PEG on immune reaction. The addition of 0.5 to 4% PEG to the diluent of biotinylated anti-SEB Ab resulted in elevated background OD readings for concentrations above 1% (Fig. 5). Because PEG improved the detection of the positive control, a concentration of 1% was selected for the supplementation of the biotinylated anti-SEB Ab diluent.
840
APPL. ENVIRON. MICROBIOL.
MORISSETTE ET AL.
TABLE 3. Saturation level and immunoreactivity of blocked CAb-coated dipsticks Peroxidase binding (OD value) (n 6)
Dipsticks
Controls Untreated GA treated
1.309 ± 0.078 0.898 ± 0.120
CAb-coated dipsticks (GA)C Unblocked Blocked with 1% lysine-1% HSAd Blocked with 1% BSA Blocked with 1% HSAd Blocked with 1% gelatin
1.018 0.318 0.138 0.291 0.325
± ± + ± ±
0.104 0.090 0.052 0.057 0.048
Detection of SEB (OD value) (n = 4) Neg
0.001 0.004
0.125 0.119 0.165 0.141 0.103
0.019 0.012 0.023 0.007 0.009
± ± ± ± ±
Index of
Pos Cb
C'
~ ~ ~ ~ ~ ~ ~ discrimination
(%
0.001 0.006 0.436 0.470 0.557 0.473 0.409
± ± ± ± ±
0.112 0.079 0.029 0.031 0.030
249 295 238 235 297
a Negative control: laboratory diluent without SEB. b Positive control: laboratory diluent with 100 ng of SEB per ml. c GA, Glutaraldehyde fixation method. d HSA, Human serum albumin.
Varying the PEG concentration in the sample diluent did not affect the discrimination between positive and negative controls, while addition of 4% PEG was required to obtain a
reaction with the streptavidin-biotin-horseradish peroxidase complex in a 5-min incubation period (results not shown). Selection of detergents for food homogenates. Of 14 detergents tested for their ability to solubilize milk fat, only 3 were able to restore the OD readings necessary for the detection of SEB in cheddar cheese homogenate (higher than 0.4) (Table 5). The other detergents gave OD values equal to or greater (up to 0.3) than that of the control cheese-no detergent sample. Because of the viscosity of the cheese homogenate, a dilution factor of 3 was required for the homogenization, bringing the probable final concentration of SEB in the liquid phase of the homogenate to approximately 66 ng/ml. The OD readings obtained with a 1.6% (wt/vol) solution of these three best detergents correspond approximately to two-thirds of the OD readings obtained with the positive controls (no cheese-no detergent) and containing 100 ng of SEB per ml. Mega-9 and Zwittergent 3-10 and 3-12 detergents were successful in eliminating the nonspecific reactions with the CAb-coated dipstick. DISCUSSION The reactivity of Abs against immobilized and/or soluble Ags is of primary importance in the development of rapid immunochemical tests for the food industry. In this work, a rapid sandwich ELISA was developed on the basis of the high reactivity of an anti-SEB Ab. This Ab gave higher reactivity toward the immobilized Ag than toward the soluble Ag (results not shown). The selection of this avid anti-SEB Ab and the optimization of the rapid sandwich
ELISA procedure allowed the detection of 0.5 ng of SEB per ml within 45 min. Immobilization of CAb. Most proteins adsorb to plastic surfaces very likely through hydrophobic interactions rather than covalent binding. Adsorbed proteins may undergo denaturation and lose immunological activity (8). Adsorption of proteins to polystyrene is concentration dependent up to a saturation limit and can result in substantial loss of antigenic or Ab activity. Desorption is also observed at a steady rate and can negatively influence the performance of the immunoassay (4). Since desorption rates as high as 40% were observed with Ab adsorbed to plastic in the presence of serum samples (7), food samples are likely to show major variations due to their highly complex nature (protein and fat composition and concentration and various other compounds). To standardize and optimize the immunoreactivity of the immobilized Abs, a conventional adsorption fixation method and two glutaraldehyde fixation procedures were evaluated. A higher concentration of CAbs was immobilized on polystyrene dipsticks by glutaraldehyde pretreatment than by conventional adsorption or combined adsorption-glutaraldehyde fixation procedures. Dobbins Place and Schroeder (7) reported that the glutaraldehyde pretreatment improved Ab retention on polystyrene. The Ag capture capacity of the immobilized Ab is critical for the performance of the assay (5). In this study, the immobilization of anti-SEB Ab on dipsticks was improved through glutaraldehyde fixation, but no attempts were made to measure concentrations of immobilized Abs nor to estimate adsorption and desorption phenomena. PEG supplementation. The enhancing effect of polymers on immunological precipitation reactions has been reported
TABLE 4. Effect of incubation time and sequence on the detection level of SEB Incubation periods
OD value of
(min)a
negative control
10/10/5/5 (n = 6) 10/10/5/10 (n = 2) 20/10/5/5 (n = 4) 20/10/5/10 (n = 2)
0.060 0.078 0.068 0.098
± ± ± ±
0.011 0.005 0.007 0.012
OD value at concn of SEB (ng/ml) of: 10 50
0.5
1.0
0.074 0.075
Q,Q92b
Q.089 0.121
0.115 0.117 0.156
0.221 0.307 0.276 0399
0.421 0.594 0.538 0.646
100
0.546 0.727 0.58
0.718
a Periods correspond to the incubation time with Ag-containing sample/biotinylated Ab conjugate/streptavidin-biotinylated enzyme complex/enzyme substrate. b Underlined values indicate OD readings equal to or higher than the negative control plus 2 standard deviations.
Q~ ~
VOL. 57,
1991
RAPID DETECTION OF SEB IN CHEESE
msuv cuo . _ o_ 8pgCAbff
0.7 E
0.6
J
lzgCAM
Positive control 0 5g
40
°M
Z0.3
0.3
~~~~~~~~~~~~~~~#.10
0.2
0.1
1
0
2
3
4
CONCENTRAMON OF PEG (%) FIG. 5. Influence of increasing concentrations of PEG in biotinylated anti-SEB Ab conjugate diluent over the Ag-Ab reaction. Immunoreactivity of CAb-coated dipsticks, coated with 8- and 10-,ug CAb concentrations without blocking treatment, was evaluated by discriminating between a positive control (diluent with 100 ng of SEB per ml) and a negative control (diluent) by using the rapid sandwich ELISA (arithmetic mean of OD values from a test performed in duplicate).
(15, 29). In the latter study, inclusion of PEG (4% [wt/vol]) in the conjugate diluent enhanced the reaction between the conjugate and the Ag-bound Ab about threefold, both at room temperature and at 37°C. When PEG was present, a 1-h incubation period at room temperature gave OD values TABLE 5. Effect of detergents on the detection of SEB in cheddar cheese samples
Sample (n = 2)
No cheese-no detergent Diluenta Neg Cb Pos Cc Laboratory diluentd Neg Cb Pos C' Cheese-no detergent Diluenta
Contamination
0.076 0.748 0.081 0.754
_e
0.031 0.178 0.019 0.141 0.081 0.543 0.082
0.031 0.100 0.026
0.004 0.168 0.041
-
0.449 0.051
0.143 0.028
0.171 0.012
+
0.410
0.134
0.181
+f Laboratory diluentd
+
Cheese + Mega-9
+
Cheese + Zwittergent
OD value at concn of detergent (wt/vol) of: 1.6% 0.16% 0.016%
-
(3-10 detergent) +
Cheese + Zwittergent
(3-12 detergent) a Diluent without Tween 20 consisted of PBS + 0.02% NaN3 + 0.1% BSA 4% PEG. b Negative control consisted of diluent without SEB. Positive control consisted of diluent with 100 ng of SEB per ml. d Laboratory diluent consisted of PBS + 0.02% NaN3 + 0.1% BSA + 0.05% Tween 20 + 4% PEG. eWithout SEB. f Containing 66 ng of SEB per ml of food homogenate. +
841
close to those obtained overnight without PEG (29). Other polymers such as dextran and polyvinyl chloride exert similar promoting effects on the interaction between soluble Ags and Abs (16). This effect is somewhat related to steric exclusion of the immune complex from the domain of the polymer (28). The concentration of PEG added to the reagents may vary according to the Ag-Ab system used. A sandwich technique was greatly improved by the addition of 2% PEG to the reagents; a sixfold shortening of the incubation time was reported by Katnik et al. (17) with no loss in either sensitivity or accuracy. In our assay, the addition of 4% PEG to the diluent of biotinylated anti-SEB Ab increased the background OD values, and no evidence could be obtained that concentrations higher than 1% could improve the sensitivity of the detection of SEB. Effect of detergents. The extraction and concentration of SE from the food sample is an essential and laborious step in any enzyme immunoassay (9, 11, 19, 23, 37). Since foods are complex systems where hydrophobic interactions are common (lipid-lipid, protein-protein, lipid-protein), detergents are likely to help minimize interference by these constituents reacting with the immunoreagents. Nonionic detergents can thus be added to prevent hydrophobic interactions between added proteins and the solid phase but without disrupting the fixation of the Ag or Ab previously adsorbed to plastic surfaces (6). The concentration of detergent needed to overcome hydrophobic interactions in aqueous systems depends on the critical micellar concentration, the micelle size, the temperature, the nature of the system, and the detergent (14). In aqueous solutions, detergent molecules occur in the form of monomers and micelles. The micelles are fairly monodisperse compact aggregates where apolar groups are sequestered in the center and polar groups face outward. The major extraction problem that was observed in our test system was the adhesion of fatty material to the CAbcoated dipsticks. After testing a full range of detergents (Table 1), one nonionic (Mega-9), and two zwitterionic (Zwittergent 3-10 and 3-12 detergents) detergents were shown to restore the Ag-Ab reaction at the level of the positive control in the absence of cheese and detergents. Further studies need to be undertaken with other foods to establish optimum detergent concentrations to be used to maximize the sensitivity of the test. Sensitivity. The double-antibody sandwich ELISA developed by Freed et al. (11) allowed the detection of less than 1 ng of SE per g of sample in several types of foods. The detection procedure could be completed within 3.5 h and necessitated at least 45 min of food preparation. By using this sandwich ELISA and two highly effective murine monoclonal Abs, Thompson et al. (34) found that the monoclonal and the polyclonal Ab system compared favorably in the detection of 1.0 ng of SE per ml. Lapeyre et al. (20) developed an indirect double sandwich ELISA by using a pair of Abs (monospecific monoclonal Ab as the CAb and polyspecific rabbit Abs as the detection system) to detect 1 ng of SE per ml within 3 h. The rapid sandwich ELISA that is described in this work gives a detection limit close to what is reported by Freed et al. (11) and Lapeyre et al. (20) but within a very short period of time (45 min). Indeed, the whole procedure for the analysis of a food sample can be completed within 1 h. This sandwich ELISA is thus a rapid and effective screening method for the detection of SEB in cheese samples, and such
842
MORISSETTE ET AL.
a test could be used advantageously by food industries for the detection of SEs in foods. ACKNOWLEDGMENTS The present study was supported by a grant from the Conseil des recherches et services agricoles du Quebec (grant IAF-84-B-1105). REFERENCES 1. Berdal, B. P., 0. Olsvik, and T. Omland. 1981. A sandwich ELISA method for detection of Staphylococcus aureus enterotoxins. Acta Pathol. Microbiol. Scand. Sect. B 89:411-415. 2. Bergdoll, M. S. 1970. Enterotoxins, p. 265-326. In T. C. Montie, S. Kadis, and S. J. AjI (ed.), Microbial toxins, vol. 3. Academic Press, Inc., London. 3. Bergdoll, M. S., R. N. Robbins, K. Weiss, C. R. Borja, I.-Y. Huang, and F. S. Chu. 1973. The staphylococcal enterotoxins: similarities. Contrib. Microbiol. Immunol. 1:390-396. 4. Butler, J. E., J. H. Peterman, M. Suter, and S. E. Dierks. 1987. The immunochemistry of solid-phase sandwich enzyme-linked immunosorbent assays. Fed. Proc. 46:2548-2556. 5. Butler, J. E., J. E. Spradling, M. Suter, S. E. Dierks, H. Heyermann, and J. H. Peterman. 1986. The immunochemistry of sandwich ELISAs. I. The binding characteristics of immunoglobulins to monoclonal and polyclonal capture antibodies adsorbed on plastic and their detection by symmetrical and asymmetrical antibody-enzyme conjugates. Mol. Immunol. 23: 971-982. 6. Clark, B. R., and E. Engvall. 1980. Enzyme-linked immunosorbent assay (ELISA): theoretical and practical aspects, p. 167179. In E. T. Maggio (ed.), Enzyme-immunoassay, chapter 8. CRC Press, Boca Raton, Fla. 7. Dobbins Place, J., and H. R. Schroeder. 1982. The fixation of anti-HBsAg on plastic surfaces. J. Immunol. Methods 48:251260. 8. Engvall, E. 1980. Enzyme immunoassay ELISA and EMIT. Methods Enzymol. 70:419-439. 9. Fey, H., H. Pfister, and 0. Ruegg. 1984. Comparative evaluation of different enzyme-linked immunosorbent assay systems for the detection of staphylococcal enterotoxins A, B, C, and D. J. Clin. Microbiol. 19:34-38. 10. Fey, H., and G. Stiffier-Rosenberg. 1977. Detection of staphylococcal enterotoxin B with a new modification of the enzyme linked immunoassay (ELISA). Experientia 33:1678. 11. Freed, R. C., M. L. Evenson, R. F. Reiser, and M. S. Bergdoll. 1982. Enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxins in foods. Appl. Environ. Microbiol. 44:1349-1355. 12. Gardas, A., and A. Lewartowska. 1988. Coating of proteins to polystyrene ELISA plates in the presence of detergents. J. Immunol. Methods 106:251-255. 13. Hahn, I. F., P. Pickenhahn, W. Lenz, and H. Brandis. 1986. An avidin-biotin ELISA for the detection of staphylococcal enterotoxins A and B. J. Immunol. Methods 92:25-29. 14. Helenius, A., D. R. McCaslin, E. Fries, and C. Tanford. 1979. Properties of detergents. Methods Enzymol. 56:734-749. 15. Hellsing, K. 1972. Letter to the editor. Immunochemistry 9:753. 16. Hellsing, K., and W. Richter. 1974. Immunochemical quantitation of dextran by a polymer enhanced nephelometric procedure. J. Immunol. Methods 5:147-151. 17. Katnik, I., M. Podg6rska, and W. Dobryszycka. 1987. Polyethylene glycol enzyme immunoassay for screening anti-haptoglobin monoclonal antibodies. J. Immunol. Methods 102:279-282. 18. Kauffman, P. E. 1980. Enzyme immunoassay for staphylococcal enterotoxin A. J. Assoc. Off. Anal. Chem. 63:1138-1143. 19. Koper, J. W., A. M. Hagenaars, and S. Notermans. 1980. Prevention of cross-reactions in the enzyme linked immunosorbent assay (ELISA) for the detection of Staphylococcus aureus enterotoxin type B in culture filtrates and foods. J. Food Safety 2:35-45. 20. Lapeyre, C., S. V. Kaveri, F. Janin, and A. D. Strosberg. 1987. Production and characterization of monoclonal antibodies to
APPL. ENVIRON. MICROBIOL.
staphylococcal enterotoxins: use in immunodetection and immunopurification. Mol. Immunol. 24:1243-1254. 21. Morissette, C., J. Goulet, and G. Lamoureux. 1990. Production of avid rabbit antibodies against staphylococcal enterotoxins A and B. J. Food Prot. 53:782-785. 22. Munoz, C., A. Nieto, A. Gaya, J. Martinez, and J. Vives. 1986. New experimental criteria for optimization of solid-phase antigen concentration and stability in ELISA. J. Immunol. Methods 94:137-144. 23. Notermans, S., R. Boot, P. D. Tips, and M. D. De NooiJ. 1983. Extraction of staphylococcal enterotoxins (SE) from minced meat and subsequent detection of SE with enzyme-linked immunosorbent assay (ELISA). J. Food Prot. 46:238-241. 24. Notermans, S., H. L. Verjans, J. Bol, and M. van Schothorst. 1978. Enzyme linked immunosorbent assay (ELISA) for determination of Staphylococcus aureus enterotoxin type B. Health Lab. Sci. 15:28-31. 25. OMS Serie de Rapports techniques. 1976. Aspects microbiologiques de l'hygiene des denrdes alimentaires. No. 598. Organisation mondiale de la Sante, Geneva. 26. Peterkin, P. I., and A. N. Sharpe. 1984. Rapid enumeration of Staphylococcus aureus in foods by direct demonstration of enterotoxigenic colonies on membrane filters by enzyme immunoassay. Appl. Environ. Microbiol. 47:1047-1053. 27. Porstmann, T., B. Porstmann, R. Wietschke, R. von Baehr, and E. Egger. 1985. Stabilization of the substrate reaction of horseradish peroxidase with 0-phenylenediamine in the enzyme immunoassay. J. Clin. Chem. Clin. Biochem. 23:41-44. 28. Rampling, M. W. 1974. The solubility of fibrinogen in solutions containing dextrans of various molecular weights. Biochem. J. 143:767-769. 29. Salonen, E.-M., and A. Vaheri. 1981. Rapid solid-phase enzyme immunoassay for antibodies to viruses and other microbes: effects of polyethylene glycol. J. Immunol. Methods 41:95-103. 30. Saunders, G. C., and M. L. Bartlett. 1977. Double-antibody solid-phase enzyme immunoassay for the detection of staphylococcal enterotoxin A. Appl. Environ. Microbiol. 34:518-522. 31. Saunders, G. C., and E. H. Clinard. 1976. Rapid micromethod of screening for antibodies to disease agents using the indirect enzyme-labeled antibody test. J. Clin. Microbiol. 3:604-608. 32. Stiffier-Rosenberg, G., and H. Fey. 1978. Simple assay for staphylococcal enterotoxins A, B, and C: modification of enzyme-linked immunosorbent assay. J. Clin. Microbiol. 8:473479. 33. Suter, M. 1982. A modified ELISA technique for anti-hapten antibodies. J. Immunol. Methods 53:103-108. 34. Thompson, N. E., M. Razdan, G. Kuntsmann, J. M. Aschenbach, M. L. Evenson, and M. S. Bergdoll. 1986. Detection of staphylococcal enterotoxins by enzyme-linked immunosorbent assays and radioimmunoassays: comparison of monoclonal and polyclonal antibody systems. Appl. Environ. Microbiol. 51:885890. 35. Tijssen, P. 1985. Practice and theory of enzyme immunoassays, p. 297-328. In R. H. Burdon and P. H. van Knippenberg (ed.), Laboratory techniques in biochemistry and molecular biology, vol. 15. Elsevier, Amsterdam. 36. Todd, E. C. D. 1983. Foodborne disease in Canada-a 5-year summary. J. Food Prot. 46:650-657. 37. Wieneke, A. A., and R. J. Gilbert. 1985. The use of a sandwich ELISA for the detection of staphylococcal enterotoxin A in foods from outbreaks of food poisoning. J. Hyg. 95:131-138. 38. Windemann, H., and E. Baumgartner. 1985. Application of enzyme-linked immunosorbent assay (ELISA) with labeled antigen for detection of staphylococcal enterotoxins A, B and C in foods. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe B 181:320-344. 39. Windemann, H., and E. Baumgartner. 1985. Application of sandwich-ELISA with labelled antibody for detection of staphylococcal enterotoxins A, B, C and D in foods. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe B 181:345-363.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1991, p. 836-842
0099-2240/91/030836-07$02.00/0 Copyright © 1991, American Society for Microbiology
Rapid and Sensitive Sandwich Enzyme-Linked Immunosorbent Assay for Detection of Staphylococcal Enterotoxin B in Cheese CELINE MORISSETTE,1 2t* JACQUES GOULET,2 AND GILLES LAMOUREUX' Centre de Recherche en Immunologie, Institut Armand-Frappier, Laval, Quebec H7V 1B7,1 and Departement de Sciences et
Technologie des Aliments, Pavillon Paul-Comtois, Universite Laval, Sainte-Foy, Quebec GIK 7P4, Canada Received 25 June 1990/Accepted 10 December 1990
A rapid and sensitive screening sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of staphylococcal enterotoxin B (SEB) in cheese by using a highly avid anti-SEB antibody (Ab) as the capture Ab (CAb) and as the biotinylated Ab conjugate. The glutaraldehyde fixation method for the immobilization of CAb on polystyrene dipsticks was superior to the adsorption fixation and the adsorptionglutaraldehyde fixation methods. The glutaraldehyde fixation method resulted in a higher surface-saturating CAb concentration as evaluated by the peroxidase saturation technique and by the ability of the CAb-coated dipstick to discriminate between positive and negative controls (index of discrimination). Of nine blocking agents used alone or in pairs, lysine-human serum albumin, bovine serum albumin, human serum albumin, and gelatin effectively saturated available sites on the CAb-coated dipsticks without causing interference with the antigen-Ab reactions. The addition of 1% polyethylene glycol to the diluent of the biotinylated anti-SEB Ab conjugate improved the detection of SEB. A concentration of 4% polyethylene glycol allowed a 5-min reaction time for the streptavidin-biotin-horseradish peroxidase conjugate. Cheddar cheese homogenate reduced the sensitivity of the SEB assay; however, the sensitivity was restored when 1.6% (wt/vol) of either a nonionic detergent (Mega-9) or two zwitterionic detergents (Zwittergent 3-10 and 3-12 detergent) was added to the diluent. By using the rapid sandwich ELISA, a minimum of 0.5 to 1.0 ng of SEB per ml was detected within 45 min. The whole procedure for the analysis of the cheddar cheese samples was completed within 1 h. This sandwich ELISA could be a rapid and sensitive screening method for the detection of staphylococcal enterotoxins in food for the agri-food industries.
Staphylococcal enterotoxins (SE) are one of the leading causes of bacterial food poisoning in Canada (36). These toxins in the active state are resistant to both proteolytic enzymes, such as trypsin, chymotrypsin, rennin, and papain (2), and high temperatures (boiling crude solutions for 30 min) (3). They have been found in organic raw materials as well as pasteurized, cooked, or otherwise processed foods (25). Rapid screening of SE for quality control in the food industry relies on rapid and sensitive methods. Many enzyme immunoassay techniques have been described for the detection of SE in culture supernatants and in food samples (1, 9-11, 13, 18, 24, 26, 30, 32, 37-39). The enzyme-linked immunosorbent assay (ELISA) is as efficient and sensitive as the radioimmunoassay but does not require the use of radioactive materials. Four types of ELISA were studied for their ability to detect SE in food samples (9). Whereas the competitive ELISA showed a higher specificity and was not sensitive to protein A in food extracts, the sandwich ELISA with labeled antibody (Ab) gave the best overall performance. Sandwich ELISAs are commonly performed by immobilizing a capture Ab (CAb) on plastic supports. The antigen (Ag) captured by the CAb support may be detected either by an enzyme-labeled Ab specific for the same determinant as the CAb or by an enzyme-labeled Ab recognizing a different epitope on the captured, multivalent Ag (4, 5). Effective pairs of monoclonal Abs and polyclonal Abs can be used in the sandwich ELISA to detect SE (20, 34). The performance
of the ELISA depends on the apparent affinity (avidity) and specificity of the selected Ab. This article describes the development of a sandwich ELISA to be used as a rapid screening assay for the detection of SE in food. By using avid polyclonal Abs to staphylococcal enterotoxin B (SEB) and a biotin-streptavidin amplification system, each major step of the analytical procedure (immobilization method of CAb, diluent for reagents, incubation period, and diluent for food preparation) was optimized and standardized to improve the sensitivity and the reaction rate of the test. MATERIALS AND METHODS SEB and Ab to SEB. SEB (Sigma Chemical Co., St. Louis, Mo.; lots 32F-4024 and 94F-4001) was used as the Ag. The preparation was rehydrated in sterile distilled water at a concentration of 1.5 mg of SEB per ml. Rabbit anti-SEB Ab containing the crude immunoglobulin fraction of antiserum was purchased from Sigma (lot 91F-40171). Diluents. Phosphate-buffered saline (0.01 M sodium phosphate-0.15 M NaCl, pH 7.4) (PBS) containing 0.02% sodium azide (NaN3) and supplemented with 0.05% Tween 20, 0.1% bovine serum albumin (BSA), and 4% polyethylene glycol 6000 (PEG) was used as a general diluent. In some experiments, the concentration of PEG was varied. Physiological saline containing 0.9% NaCl, 0.05% Tween 20, and 0.01% NaN3 was used as a washing solution. Biotinylated Abs. The anti-SEB Ab was biotinylated by use of the procedure of GIBCO-BRL (Burlington, Ontario, Canada). Biotin-N-hydroxysuccinimide ester (Bethesda Research Laboratories/Life Technologies, Inc., Gaithersburg, Md.; lot 51101) was dissolved to a concentration of 50 mg/ml
* Corresponding author. t Present address: Food Research Centre, Research Branch, Agriculture Canada, Central Experimental Farm, Ottawa, Ontario
KlA 0C6, Canada.
836
VOL . 57 i
RAPID DETECTION OF SEB IN CHEESE
1991
TABLE 1. Molecular weight, critical micellar concentration, and ionic state of detergents testeda Mol wt
Critical micellar concn (M)
Hexyl-p-D-glucopyranoside
264.3
Large
Nonyl-p-D-glucopyranoside
306.4 510.6
Detergent
Ionic state
Detergent test kit
Heptyl-3-D-glucopyranoside 278.3 Octyl-f3-D-glucopyranoside 292.4 Dodecyl-,B-D-maltoside Mega test kit Mega-8 Mega-9
321.5 335.5
Zwittergent test kit Zwittergent 3-08 detergent Zwittergent 3-10 detergent Zwittergent 3-12 detergent Zwittergent 3-14 detergent
279.6 307.6 335.6 363.6
Other common detergents Sodium dodecyl sulfate Tween 20 Dishwashing liquid (Ivory)
288.4
2.5 x 10-2 6.5 x 10-3
Nonionic Nonionic Nonionic Nonionic Nonionic Nonionic Nonionic
Large 3.9 x 10-2 3.6 x 10-3 3.3 x 10-4
Zwitterionic Zwitterionic Zwitterionic
5.9 x 10-5
Ionic Nonionic
Zwitterionic
a From Calbiochem Biochemical & Immunochemical catalog 87, Helenius et al. (14), and Gardas and Lewartowska (12).
in dry N,N-dimethyl formamide (Fisher Scientific Co., Orangeburg, N.Y.; catalog no. D-119). A 10-pul volume of this solution was reacted with 0.125 ml of protein (20 mg/ml), 20 p.l of 0.5 M NaHCO3 (pH 9.0), and 40 p.l of H20 for 1 h at room temperature with gentle agitation. The reaction was stopped by adding 25 p. of 1 M NH4Cl. The mixture was dialyzed against PBS and membrane filtered (pore size, 0.45
Am).
A biotinylated donkey anti-rabbit immunoglobulin (Amersham Canada Ltd., Oakville, Ontario, Canada; catalog no. RPN 1004, lot 30) was also used to detect the presence of rabbit Abs on dipsticks. Streptavidin-biotinylated enzyme complex. Biotinylated anti-rabbit immunoglobulin and biotinylated anti-SEB Ab were detected by a preformed streptavidin-biotinylated horseradish peroxidase complex from Amersham Canada (catalog no. RPN 1051, lot 42 and 61). Enzyme substrate. A horseradish peroxidase substrate solution was freshly prepared by dissolving 100 mg of o-diaminobenzene dihydrochloride (Sigma) in 100 ml of 0.2 M Na2-phosphate-0.1 M citrate buffer, pH 5, containing 150 p.l of 30% H202. The peroxidase reaction was stopped by the addition of 0.5 ml of a solution containing 2 M H2SO4 and 0.1 M NaSO3 (27). Detergents. Several detergents (Table 1) were used as substitutes for Tween 20 in the diluent for the preparation of food homogenates. All selected detergents were soluble in PBS-NaN3-BSA-4% PEG. The Detergent Test Kit, Mega Test Kit, and Zwittergent Test Kit were obtained from Calbiochem Biochemical & Immunochemical (Terochem Laboratories Ltd., Edmonton, Alberta, Canada). Sodium dodecyl sulfate was obtained from Matheson Coleman & Bell, Norwood, Ohio, and Tween 20, polyoxyethylene sorbitan monolaureate, was obtained from Fisher Scientific. Immobilization of CAbs on polystyrene dipsticks. (i) Adsorption fixation method. A batch of 500 polystyrene dipsticks (Sarstedt no. 81,970, Ruhr, West Germany) was washed with deionized water for 2 h and incubated overnight
837
in a 0.01 M carbonate buffer (pH 9.6) containing increasing concentrations (0, 0.5, 1, 5, 10, and 25 p.g/ml) of CAb. In some experiments, the remaining active sites on the CAbcoated dipsticks were saturated for 2 h with a solution of blocking agent (1% BSA in PBS). All incubations were performed at room temperature (22°C) with agitation on a rotary shaker. After two washings with PBS-NaN3-Tween 20, the CAb-coated dipsticks were stored in PBS-NaN3Tween 20 at 4°C until required. (ii) Combined adsorption-glutaraldehyde fixation method. The adsorption-glutaraldehyde fixation method was adapted from the binding technique of Saunders and Clinard (31). A batch of 500 polystyrene dipsticks previously washed with deionized water for 2 h was incubated for 1 h in a 0.01 M carbonate buffer (pH 9.6) containing increasing concentrations (0, 0.5, 1, 5, 10, and 25 pug/ml) of CAb. The CAb-coated dipsticks were then incubated with 0.25% (vol/vol) glutaraldehyde in PBS (pH 7.0) for 30 min, washed four times with distilled water, and reincubated overnight in the same CAbcontaining solutions. In some experiments, the remaining active sites of the CAb-coated dipsticks were saturated with a solution of blocking agent (1% BSA in PBS) for 2 h. All incubations were performed at room temperature (22°C) with agitation on a rotary shaker. After two washings with PBS-NaN3-Tween 20, the CAb-coated dipsticks were stored in PBS-NaN3-Tween 20 at 4°C until required. (iii) Glutaraldehyde fixation method. The glutaraldehyde fixation method was adapted from the method of Suter (33) by the method of Tijssen (35). A batch of 500 polystyrene dipsticks was washed twice with distilled water and once with 0.1 M sodium phosphate buffer (pH 5). The dipsticks were pretreated with 0.2% glutaraldehyde (Sigma; catalog no. G-6257, lot 116F-5027, grade II) in 0.1 M sodium phosphate buffer (pH 5) for 4 h. The dipsticks were washed three times with the same buffer and incubated in 0.1 M sodium phosphate buffer (pH 8) containing increasing concentrations (0, 2, 4, 8, 10, 20, 40, and 60 p.g/ml) of CAb for 14 to 16 h. The CAb-coated dipsticks were then washed three times with PBS (pH 7.4), and in some experiments the non-Ab-occupied sites were saturated by incubating dipsticks for 2 h in a solution of blocking agent (100 mM lysine) in PBS. All incubations were performed at room temperature (22°C) with agitation on a rotary shaker. After two washings with PBS (pH 7.4), the dipsticks were stored in PBS-NaN3Tween 20 at 4°C until required. Rapid sandwich ELISA. In a typical assay (Fig. 1), a CAb-coated dipstick was incubated with 1.0 ml of SEcontaminated sample for 5 min. All incubation steps were performed at room temperature with agitation. After two washings with the washing solution, the CAb-coated dipstick was transferred to a tube containing 300 p.1 of a dilution of biotinylated anti-SE Ab conjugates. After 5 min of incubation, the dipstick was washed twice and transferred to a tube containing 300 pul of diluted streptavidin-biotin-horseradish peroxidase complex. The CAb-coated dipstick was removed after 5 min, washed twice, and transferred to a tube containing 1.0 ml of H202-o-diaminobenzene dihydrochloride for 5 min. After removing the dipstick, the reaction was stopped by transferring the reaction mixture into tubes containing the stopping solution. The optical density of the solution at 490 nm (OD49) was read with an optical fiber probe (PC 800; Brinkmann [Canada Ltd.], Rexdale, Ontario, Canada). Blocking treatment. Several reagents (Table 2) were tested for their ability to block nonspecific binding and saturate the unoccupied sites on surface-immobilized CAb. They were used alone or in pairs at a concentration of 1% in PBS. After
,.
_A
APPL. ENVIRON. MICROBIOL.
MORISSETTE ET AL.
838
Step
1
s. .
C'0 0
.
0
Step 2
Step 3
*
A
A
TABLE 2. Reagents used as blocking agents after immobilization of Abs on polystyrene dipsticks
Sigma (catalog no. L-6001) Fisher (catalog no. G-46) albumin .........I n-house preparation (Institut
DL-Lysine ................ Glycine ................. Human serum 0
Io
Armand-Frappier) Miles Laboratories, Inc., Naperville, Ill. (catalog no. 81-066-1) Whole rabbit serum .............I n-house preparation (single bleed from one animal) Bacto-Peptone ................ Difco (catalog no. 0118-05-4) Casein ................ Sigma (catalog no. C-5890) Gelatin ................ Sigma; from calf skin type IV (catalog no. G-0510) Organosilane (Prosil-28) ....... SCM Chemicals, Gainesville, Fla. (catalog no. 11975-0) BSA .................
,S 20 minutes
10 minutes
Washing
Washing
-4
Washing
CAb-coated dipsticks was employed to discriminate between positive and negative controls. Peroxidase saturation technique. The peroxidase saturation technique of Munioz et al. (22) was used to estimate the Ab-free surface sites after the immobilization. Horseradish peroxidase (Boehringer GmbH; lot 10300224-40, 308 U/mg) diluted to 0.1 mg/ml in PBS was incubated with CAb-coated dipsticks for 2 h at room temperature with agitation. The dipsticks were then washed four times with PBS, and the enzyme activity was developed as described above. Preparation of cheese samples. Five-gram samples of cheddar cheese contaminated with 200 ng of SEB per g were homogenized in a blender for 15 s with 10 ml of diluent containing various concentrations (0.016, 0.16, and 1.6% [wt/vol]) of detergents (Table 1). A 1.0-ml aliquot of the homogenate was used for testing with the rapid sandwich ELISA. Index of discrimination. To evaluate the immunoreactivity of the CAb-coated dipsticks in the rapid sandwich ELISA, we used a discrimination index defined as follows: (OD490 of
positive control - OD490 of negative control)/OD490 of negative control x 100 = %.
_
t ': ..;
.-. A:..
:.-.
. o
OD 490
SE Antibody against SE Biotinylated anti-SE Ab conjugate A _ s_%b
Source
Reagent
_
_
t
._s2_
FIG. 1. Major steps of the rapid sandwich ELISA procedure. OPD, o-Diaminobenzene dihydrochloride.
immobilization by using an unsaturating concentration of CAb (4 ,ug/ml), the dipsticks were incubated with the blocking agents for 2 h and washed three times. The capacity of the blocking agents to saturate the Ab-free sites on CAbcoated dipsticks was evaluated by the peroxidase saturation technique. The rapid sandwich ELISA with the blocked
RESULTS Selection of Abs. The anti-SEB Ab used in this study was selected for its high avidity for SEB. The anti-SEB Ab ELISA titer of the crude immunoglobulin fraction was 409,600 at the endpoint of negative control, when incubated for a 5-min period with SEB-coated dipsticks and a dilution of the protein A-horseradish peroxidase conjugate (21). Selection of a method to immobilize the CAb. Three immobilization methods, adsorption fixation, adsorption-glutaraldehyde fixation, and glutaraldehyde fixation, were compared for their ability to bind CAb to polystyrene dipsticks under saturating conditions and for the efficiency of the resulting CAb-coated dipsticks to discriminate between negative and positive controls. After the immobilization of CAb, residual free sites were evaluated by the peroxidase saturation technique. In this technique, the OD value is directly proportional to the available binding sites on support. Accordingly, the OD values decreased as the CAb concentration increased, and regression lines for the rapidly decreasing OD readings and for the slowly decreasing (near-constant) OD readings were calculated (Fig. 2). By using the glutaraldehyde fixation method, the surface-saturating CAb concentration was evaluated as the intersection point between both regression lines (22), i.e., 11 ,ug/ml. The adsorption fixation
RAPID DETECTION OF SEB IN CHEESE
VOL. 57, 1991
0.7
900 ,2 800 O 700 > 600 2 500 a: ) 400 D 300 0 200
E
0.6 z 0.5 z < 0.4 0 0.4 00.3 0.3
0
.. .. .. .. 60 20 30 40 50 10 CONCENTRATION OF CAb (ligtmQ
FIG. 2. Immobilization of increasing concentrations of CAb with the glutaraldehyde fixation method. Non-Ab-occupied surface sites were evaluated by the peroxidase saturation technique (n = 8, arithmetic mean of OD values from a test repeated four times in duplicate).
839
GA-CAb
GA-CAb-Lysirw
v
0 100
z
0
0
10
20
30
40
50
60
CONCENTRATION OF CAb (igml) FIG. 4. Immunoreactivity of CAb-coated dipsticks as estimated by the index of discrimination. GA, Glutaraldehyde fixation method.
and adsorption-glutaraldehyde fixation methods demonstrated a surface-saturating CAb concentration of approximately 5 ,ug/ml, a twofold decrease compared with the concentration demonstrated by the glutaraldehyde fixation method. The ability to discriminate between positive and negative controls with the CAb-coated dipsticks obtained from the glutaraldehyde fixation method is represented in Fig. 3 and 4. A maximum immunoreactivity was observed at about 10 jig of CAb per ml. Results expressed as the index of discrimination allow visualization of the surface-saturating CAb concentration where the OD value of the positive control is increased while the OD value of the negative control remains low. The index of discrimination for the adsorption fixation and the adsorption-glutaraldehyde fixation methods was at best 94 and 265%, respectively, at a concentration of 5 ,ug of CAb per ml. The adsorptionglutaraldehyde fixation and the glutaraldehyde fixation methods gave good discrimination at their surface-saturating CAb concentration; however, the glutaraldehyde fixation method was preferred because of its higher index of discrimination and its higher surface-saturating CAb concentration. How1.2
Negative control GA-CAb &
E
---
GA-CAb-Lysne
010
Positive control
*
F _iir ~GACAb_
-
< 0.8
GA-CAb-Lysin
0.6 w ..j
0.4
0 0
30 40 50 20 CONCENTRATION OF CAb (1tgIml)
10
60
FIG. 3. Immunoreactivity of CAb-coated dipsticks evaluated by the detection of a positive control (laboratory diluent containing 145 ng of SEB per ml) and a negative control (laboratory diluent) by rapid sandwich ELISA (arithmetic mean of OD values from a test performed in duplicate). GA, Glutaraldehyde fixation method.
ever, when this latter method was used with the suggested blocking agent, lysine (35), interference was observed on the detection of SEB (Fig. 3 and 4). Selection of a blocking agent. Preliminary tests were conducted with a series of nine blocking agents used alone or in pairs and with dipsticks coated with 4 jig of anti-SEB Ab per ml (undersaturating CAb concentration). Table 3 shows the capacity of the four best blocking agents to inhibit the binding of the peroxidase and their influence over the immunoreactivity of the CAb-coated dipsticks in the rapid sandwich ELISA. The index of discrimination of the CAbcoated dipsticks blocked with those agents was similar to the one obtained with the unblocked controls, indicating that their effect is not detrimental to the immunological reaction in undersaturating conditions. The following other blocking agents gave either a high OD reading with the peroxidase saturation technique (do not saturate available sites) or poor index of discrimination (interfere with the further reactions): lysine, lysine-organosilane, lysine-BSA, lysine-glycine, glycine, glycine-organosilane, glycine-human serum albumin, peptone, peptone-lysine, peptone-glycine, and whole rabbit serum. At the optimal coating CAb concentration (11 ,ug of anti-SEB Ab per ml), the effect of the four best blocking agents was maintained. Because of its high index of discrimination and its low OD value for negative control, gelatin was selected as the best blocking agent for high dilutions of biotinylated anti-SEB Ab. Selection of the incubation periods. Minimal incubation periods were selected to detect the critical enterotoxin concentration (100 ng of SE per 100 g of food or 0.5 ng of SE per ml of food homogenate) assuming a dilution factor of 2. This minimum concentration was detected when the period of incubation of CAb-coated dipstick with the SEB-containing sample was longer than 10 min; for an incubation time of 20 min, the minimal detection limit is around 0.5 ng of SEB per ml (Table 4). This period of incubation can be increased when the dilution factor is higher than 2. Effect of PEG on immune reaction. The addition of 0.5 to 4% PEG to the diluent of biotinylated anti-SEB Ab resulted in elevated background OD readings for concentrations above 1% (Fig. 5). Because PEG improved the detection of the positive control, a concentration of 1% was selected for the supplementation of the biotinylated anti-SEB Ab diluent.
840
APPL. ENVIRON. MICROBIOL.
MORISSETTE ET AL.
TABLE 3. Saturation level and immunoreactivity of blocked CAb-coated dipsticks Peroxidase binding (OD value) (n 6)
Dipsticks
Controls Untreated GA treated
1.309 ± 0.078 0.898 ± 0.120
CAb-coated dipsticks (GA)C Unblocked Blocked with 1% lysine-1% HSAd Blocked with 1% BSA Blocked with 1% HSAd Blocked with 1% gelatin
1.018 0.318 0.138 0.291 0.325
± ± + ± ±
0.104 0.090 0.052 0.057 0.048
Detection of SEB (OD value) (n = 4) Neg
0.001 0.004
0.125 0.119 0.165 0.141 0.103
0.019 0.012 0.023 0.007 0.009
± ± ± ± ±
Index of
Pos Cb
C'
~ ~ ~ ~ ~ ~ ~ discrimination
(%
0.001 0.006 0.436 0.470 0.557 0.473 0.409
± ± ± ± ±
0.112 0.079 0.029 0.031 0.030
249 295 238 235 297
a Negative control: laboratory diluent without SEB. b Positive control: laboratory diluent with 100 ng of SEB per ml. c GA, Glutaraldehyde fixation method. d HSA, Human serum albumin.
Varying the PEG concentration in the sample diluent did not affect the discrimination between positive and negative controls, while addition of 4% PEG was required to obtain a
reaction with the streptavidin-biotin-horseradish peroxidase complex in a 5-min incubation period (results not shown). Selection of detergents for food homogenates. Of 14 detergents tested for their ability to solubilize milk fat, only 3 were able to restore the OD readings necessary for the detection of SEB in cheddar cheese homogenate (higher than 0.4) (Table 5). The other detergents gave OD values equal to or greater (up to 0.3) than that of the control cheese-no detergent sample. Because of the viscosity of the cheese homogenate, a dilution factor of 3 was required for the homogenization, bringing the probable final concentration of SEB in the liquid phase of the homogenate to approximately 66 ng/ml. The OD readings obtained with a 1.6% (wt/vol) solution of these three best detergents correspond approximately to two-thirds of the OD readings obtained with the positive controls (no cheese-no detergent) and containing 100 ng of SEB per ml. Mega-9 and Zwittergent 3-10 and 3-12 detergents were successful in eliminating the nonspecific reactions with the CAb-coated dipstick. DISCUSSION The reactivity of Abs against immobilized and/or soluble Ags is of primary importance in the development of rapid immunochemical tests for the food industry. In this work, a rapid sandwich ELISA was developed on the basis of the high reactivity of an anti-SEB Ab. This Ab gave higher reactivity toward the immobilized Ag than toward the soluble Ag (results not shown). The selection of this avid anti-SEB Ab and the optimization of the rapid sandwich
ELISA procedure allowed the detection of 0.5 ng of SEB per ml within 45 min. Immobilization of CAb. Most proteins adsorb to plastic surfaces very likely through hydrophobic interactions rather than covalent binding. Adsorbed proteins may undergo denaturation and lose immunological activity (8). Adsorption of proteins to polystyrene is concentration dependent up to a saturation limit and can result in substantial loss of antigenic or Ab activity. Desorption is also observed at a steady rate and can negatively influence the performance of the immunoassay (4). Since desorption rates as high as 40% were observed with Ab adsorbed to plastic in the presence of serum samples (7), food samples are likely to show major variations due to their highly complex nature (protein and fat composition and concentration and various other compounds). To standardize and optimize the immunoreactivity of the immobilized Abs, a conventional adsorption fixation method and two glutaraldehyde fixation procedures were evaluated. A higher concentration of CAbs was immobilized on polystyrene dipsticks by glutaraldehyde pretreatment than by conventional adsorption or combined adsorption-glutaraldehyde fixation procedures. Dobbins Place and Schroeder (7) reported that the glutaraldehyde pretreatment improved Ab retention on polystyrene. The Ag capture capacity of the immobilized Ab is critical for the performance of the assay (5). In this study, the immobilization of anti-SEB Ab on dipsticks was improved through glutaraldehyde fixation, but no attempts were made to measure concentrations of immobilized Abs nor to estimate adsorption and desorption phenomena. PEG supplementation. The enhancing effect of polymers on immunological precipitation reactions has been reported
TABLE 4. Effect of incubation time and sequence on the detection level of SEB Incubation periods
OD value of
(min)a
negative control
10/10/5/5 (n = 6) 10/10/5/10 (n = 2) 20/10/5/5 (n = 4) 20/10/5/10 (n = 2)
0.060 0.078 0.068 0.098
± ± ± ±
0.011 0.005 0.007 0.012
OD value at concn of SEB (ng/ml) of: 10 50
0.5
1.0
0.074 0.075
Q,Q92b
Q.089 0.121
0.115 0.117 0.156
0.221 0.307 0.276 0399
0.421 0.594 0.538 0.646
100
0.546 0.727 0.58
0.718
a Periods correspond to the incubation time with Ag-containing sample/biotinylated Ab conjugate/streptavidin-biotinylated enzyme complex/enzyme substrate. b Underlined values indicate OD readings equal to or higher than the negative control plus 2 standard deviations.
Q~ ~
VOL. 57,
1991
RAPID DETECTION OF SEB IN CHEESE
msuv cuo . _ o_ 8pgCAbff
0.7 E
0.6
J
lzgCAM
Positive control 0 5g
40
°M
Z0.3
0.3
~~~~~~~~~~~~~~~#.10
0.2
0.1
1
0
2
3
4
CONCENTRAMON OF PEG (%) FIG. 5. Influence of increasing concentrations of PEG in biotinylated anti-SEB Ab conjugate diluent over the Ag-Ab reaction. Immunoreactivity of CAb-coated dipsticks, coated with 8- and 10-,ug CAb concentrations without blocking treatment, was evaluated by discriminating between a positive control (diluent with 100 ng of SEB per ml) and a negative control (diluent) by using the rapid sandwich ELISA (arithmetic mean of OD values from a test performed in duplicate).
(15, 29). In the latter study, inclusion of PEG (4% [wt/vol]) in the conjugate diluent enhanced the reaction between the conjugate and the Ag-bound Ab about threefold, both at room temperature and at 37°C. When PEG was present, a 1-h incubation period at room temperature gave OD values TABLE 5. Effect of detergents on the detection of SEB in cheddar cheese samples
Sample (n = 2)
No cheese-no detergent Diluenta Neg Cb Pos Cc Laboratory diluentd Neg Cb Pos C' Cheese-no detergent Diluenta
Contamination
0.076 0.748 0.081 0.754
_e
0.031 0.178 0.019 0.141 0.081 0.543 0.082
0.031 0.100 0.026
0.004 0.168 0.041
-
0.449 0.051
0.143 0.028
0.171 0.012
+
0.410
0.134
0.181
+f Laboratory diluentd
+
Cheese + Mega-9
+
Cheese + Zwittergent
OD value at concn of detergent (wt/vol) of: 1.6% 0.16% 0.016%
-
(3-10 detergent) +
Cheese + Zwittergent
(3-12 detergent) a Diluent without Tween 20 consisted of PBS + 0.02% NaN3 + 0.1% BSA 4% PEG. b Negative control consisted of diluent without SEB. Positive control consisted of diluent with 100 ng of SEB per ml. d Laboratory diluent consisted of PBS + 0.02% NaN3 + 0.1% BSA + 0.05% Tween 20 + 4% PEG. eWithout SEB. f Containing 66 ng of SEB per ml of food homogenate. +
841
close to those obtained overnight without PEG (29). Other polymers such as dextran and polyvinyl chloride exert similar promoting effects on the interaction between soluble Ags and Abs (16). This effect is somewhat related to steric exclusion of the immune complex from the domain of the polymer (28). The concentration of PEG added to the reagents may vary according to the Ag-Ab system used. A sandwich technique was greatly improved by the addition of 2% PEG to the reagents; a sixfold shortening of the incubation time was reported by Katnik et al. (17) with no loss in either sensitivity or accuracy. In our assay, the addition of 4% PEG to the diluent of biotinylated anti-SEB Ab increased the background OD values, and no evidence could be obtained that concentrations higher than 1% could improve the sensitivity of the detection of SEB. Effect of detergents. The extraction and concentration of SE from the food sample is an essential and laborious step in any enzyme immunoassay (9, 11, 19, 23, 37). Since foods are complex systems where hydrophobic interactions are common (lipid-lipid, protein-protein, lipid-protein), detergents are likely to help minimize interference by these constituents reacting with the immunoreagents. Nonionic detergents can thus be added to prevent hydrophobic interactions between added proteins and the solid phase but without disrupting the fixation of the Ag or Ab previously adsorbed to plastic surfaces (6). The concentration of detergent needed to overcome hydrophobic interactions in aqueous systems depends on the critical micellar concentration, the micelle size, the temperature, the nature of the system, and the detergent (14). In aqueous solutions, detergent molecules occur in the form of monomers and micelles. The micelles are fairly monodisperse compact aggregates where apolar groups are sequestered in the center and polar groups face outward. The major extraction problem that was observed in our test system was the adhesion of fatty material to the CAbcoated dipsticks. After testing a full range of detergents (Table 1), one nonionic (Mega-9), and two zwitterionic (Zwittergent 3-10 and 3-12 detergents) detergents were shown to restore the Ag-Ab reaction at the level of the positive control in the absence of cheese and detergents. Further studies need to be undertaken with other foods to establish optimum detergent concentrations to be used to maximize the sensitivity of the test. Sensitivity. The double-antibody sandwich ELISA developed by Freed et al. (11) allowed the detection of less than 1 ng of SE per g of sample in several types of foods. The detection procedure could be completed within 3.5 h and necessitated at least 45 min of food preparation. By using this sandwich ELISA and two highly effective murine monoclonal Abs, Thompson et al. (34) found that the monoclonal and the polyclonal Ab system compared favorably in the detection of 1.0 ng of SE per ml. Lapeyre et al. (20) developed an indirect double sandwich ELISA by using a pair of Abs (monospecific monoclonal Ab as the CAb and polyspecific rabbit Abs as the detection system) to detect 1 ng of SE per ml within 3 h. The rapid sandwich ELISA that is described in this work gives a detection limit close to what is reported by Freed et al. (11) and Lapeyre et al. (20) but within a very short period of time (45 min). Indeed, the whole procedure for the analysis of a food sample can be completed within 1 h. This sandwich ELISA is thus a rapid and effective screening method for the detection of SEB in cheese samples, and such
842
MORISSETTE ET AL.
a test could be used advantageously by food industries for the detection of SEs in foods. ACKNOWLEDGMENTS The present study was supported by a grant from the Conseil des recherches et services agricoles du Quebec (grant IAF-84-B-1105). REFERENCES 1. Berdal, B. P., 0. Olsvik, and T. Omland. 1981. A sandwich ELISA method for detection of Staphylococcus aureus enterotoxins. Acta Pathol. Microbiol. Scand. Sect. B 89:411-415. 2. Bergdoll, M. S. 1970. Enterotoxins, p. 265-326. In T. C. Montie, S. Kadis, and S. J. AjI (ed.), Microbial toxins, vol. 3. Academic Press, Inc., London. 3. Bergdoll, M. S., R. N. Robbins, K. Weiss, C. R. Borja, I.-Y. Huang, and F. S. Chu. 1973. The staphylococcal enterotoxins: similarities. Contrib. Microbiol. Immunol. 1:390-396. 4. Butler, J. E., J. H. Peterman, M. Suter, and S. E. Dierks. 1987. The immunochemistry of solid-phase sandwich enzyme-linked immunosorbent assays. Fed. Proc. 46:2548-2556. 5. Butler, J. E., J. E. Spradling, M. Suter, S. E. Dierks, H. Heyermann, and J. H. Peterman. 1986. The immunochemistry of sandwich ELISAs. I. The binding characteristics of immunoglobulins to monoclonal and polyclonal capture antibodies adsorbed on plastic and their detection by symmetrical and asymmetrical antibody-enzyme conjugates. Mol. Immunol. 23: 971-982. 6. Clark, B. R., and E. Engvall. 1980. Enzyme-linked immunosorbent assay (ELISA): theoretical and practical aspects, p. 167179. In E. T. Maggio (ed.), Enzyme-immunoassay, chapter 8. CRC Press, Boca Raton, Fla. 7. Dobbins Place, J., and H. R. Schroeder. 1982. The fixation of anti-HBsAg on plastic surfaces. J. Immunol. Methods 48:251260. 8. Engvall, E. 1980. Enzyme immunoassay ELISA and EMIT. Methods Enzymol. 70:419-439. 9. Fey, H., H. Pfister, and 0. Ruegg. 1984. Comparative evaluation of different enzyme-linked immunosorbent assay systems for the detection of staphylococcal enterotoxins A, B, C, and D. J. Clin. Microbiol. 19:34-38. 10. Fey, H., and G. Stiffier-Rosenberg. 1977. Detection of staphylococcal enterotoxin B with a new modification of the enzyme linked immunoassay (ELISA). Experientia 33:1678. 11. Freed, R. C., M. L. Evenson, R. F. Reiser, and M. S. Bergdoll. 1982. Enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxins in foods. Appl. Environ. Microbiol. 44:1349-1355. 12. Gardas, A., and A. Lewartowska. 1988. Coating of proteins to polystyrene ELISA plates in the presence of detergents. J. Immunol. Methods 106:251-255. 13. Hahn, I. F., P. Pickenhahn, W. Lenz, and H. Brandis. 1986. An avidin-biotin ELISA for the detection of staphylococcal enterotoxins A and B. J. Immunol. Methods 92:25-29. 14. Helenius, A., D. R. McCaslin, E. Fries, and C. Tanford. 1979. Properties of detergents. Methods Enzymol. 56:734-749. 15. Hellsing, K. 1972. Letter to the editor. Immunochemistry 9:753. 16. Hellsing, K., and W. Richter. 1974. Immunochemical quantitation of dextran by a polymer enhanced nephelometric procedure. J. Immunol. Methods 5:147-151. 17. Katnik, I., M. Podg6rska, and W. Dobryszycka. 1987. Polyethylene glycol enzyme immunoassay for screening anti-haptoglobin monoclonal antibodies. J. Immunol. Methods 102:279-282. 18. Kauffman, P. E. 1980. Enzyme immunoassay for staphylococcal enterotoxin A. J. Assoc. Off. Anal. Chem. 63:1138-1143. 19. Koper, J. W., A. M. Hagenaars, and S. Notermans. 1980. Prevention of cross-reactions in the enzyme linked immunosorbent assay (ELISA) for the detection of Staphylococcus aureus enterotoxin type B in culture filtrates and foods. J. Food Safety 2:35-45. 20. Lapeyre, C., S. V. Kaveri, F. Janin, and A. D. Strosberg. 1987. Production and characterization of monoclonal antibodies to
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staphylococcal enterotoxins: use in immunodetection and immunopurification. Mol. Immunol. 24:1243-1254. 21. Morissette, C., J. Goulet, and G. Lamoureux. 1990. Production of avid rabbit antibodies against staphylococcal enterotoxins A and B. J. Food Prot. 53:782-785. 22. Munoz, C., A. Nieto, A. Gaya, J. Martinez, and J. Vives. 1986. New experimental criteria for optimization of solid-phase antigen concentration and stability in ELISA. J. Immunol. Methods 94:137-144. 23. Notermans, S., R. Boot, P. D. Tips, and M. D. De NooiJ. 1983. Extraction of staphylococcal enterotoxins (SE) from minced meat and subsequent detection of SE with enzyme-linked immunosorbent assay (ELISA). J. Food Prot. 46:238-241. 24. Notermans, S., H. L. Verjans, J. Bol, and M. van Schothorst. 1978. Enzyme linked immunosorbent assay (ELISA) for determination of Staphylococcus aureus enterotoxin type B. Health Lab. Sci. 15:28-31. 25. OMS Serie de Rapports techniques. 1976. Aspects microbiologiques de l'hygiene des denrdes alimentaires. No. 598. Organisation mondiale de la Sante, Geneva. 26. Peterkin, P. I., and A. N. Sharpe. 1984. Rapid enumeration of Staphylococcus aureus in foods by direct demonstration of enterotoxigenic colonies on membrane filters by enzyme immunoassay. Appl. Environ. Microbiol. 47:1047-1053. 27. Porstmann, T., B. Porstmann, R. Wietschke, R. von Baehr, and E. Egger. 1985. Stabilization of the substrate reaction of horseradish peroxidase with 0-phenylenediamine in the enzyme immunoassay. J. Clin. Chem. Clin. Biochem. 23:41-44. 28. Rampling, M. W. 1974. The solubility of fibrinogen in solutions containing dextrans of various molecular weights. Biochem. J. 143:767-769. 29. Salonen, E.-M., and A. Vaheri. 1981. Rapid solid-phase enzyme immunoassay for antibodies to viruses and other microbes: effects of polyethylene glycol. J. Immunol. Methods 41:95-103. 30. Saunders, G. C., and M. L. Bartlett. 1977. Double-antibody solid-phase enzyme immunoassay for the detection of staphylococcal enterotoxin A. Appl. Environ. Microbiol. 34:518-522. 31. Saunders, G. C., and E. H. Clinard. 1976. Rapid micromethod of screening for antibodies to disease agents using the indirect enzyme-labeled antibody test. J. Clin. Microbiol. 3:604-608. 32. Stiffier-Rosenberg, G., and H. Fey. 1978. Simple assay for staphylococcal enterotoxins A, B, and C: modification of enzyme-linked immunosorbent assay. J. Clin. Microbiol. 8:473479. 33. Suter, M. 1982. A modified ELISA technique for anti-hapten antibodies. J. Immunol. Methods 53:103-108. 34. Thompson, N. E., M. Razdan, G. Kuntsmann, J. M. Aschenbach, M. L. Evenson, and M. S. Bergdoll. 1986. Detection of staphylococcal enterotoxins by enzyme-linked immunosorbent assays and radioimmunoassays: comparison of monoclonal and polyclonal antibody systems. Appl. Environ. Microbiol. 51:885890. 35. Tijssen, P. 1985. Practice and theory of enzyme immunoassays, p. 297-328. In R. H. Burdon and P. H. van Knippenberg (ed.), Laboratory techniques in biochemistry and molecular biology, vol. 15. Elsevier, Amsterdam. 36. Todd, E. C. D. 1983. Foodborne disease in Canada-a 5-year summary. J. Food Prot. 46:650-657. 37. Wieneke, A. A., and R. J. Gilbert. 1985. The use of a sandwich ELISA for the detection of staphylococcal enterotoxin A in foods from outbreaks of food poisoning. J. Hyg. 95:131-138. 38. Windemann, H., and E. Baumgartner. 1985. Application of enzyme-linked immunosorbent assay (ELISA) with labeled antigen for detection of staphylococcal enterotoxins A, B and C in foods. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe B 181:320-344. 39. Windemann, H., and E. Baumgartner. 1985. Application of sandwich-ELISA with labelled antibody for detection of staphylococcal enterotoxins A, B, C and D in foods. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe B 181:345-363.
