APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1977, Copyright X) 1977 American Society for Microbiology
p. 947-954
Vol. 33, No. 4 Printed in U.S.A.
Use of Enzyme-Labeled Antibodies to Detect Salmonella in Foods1 E. P. KRYSINSKI2 AND R. C. HEIMSCH* Department of Bacteriology and Biochemistry, University ofIdaho, Moscow, Idaho 83843
Received for publication 29 November 1976
An indirect enzyme-labeled antibody technique (ELAT), in which Salmonella typhimurium was used as a model, was developed as a method to detect Salmonella in food samples. A cellulose-acetate membrane filter, the matrix for detection, was placed on a membrane-filter base and overlaid with a multiwelled lucite template. Mixed broth enrichment cultures were dispensed in the template wells, and cells were spotted onto the membrane via suction. After fixation, the membranes were immersed in rabbit anti-S. typhimurium flagella antibody, washed, immersed in goat anti-rabbit antibody conjugated to peroxidase, and washed. Exposure of membranes to the substrates 3,3'-diaminobenzidine or benzidine resulted in development of brown or blue macroscopic reaction products, respectively, on spots containing S. typhimurium. ELAT results agreed with those of enrichment serology and cultural procedures on three food products containing known levels of S. typhimurium. Because of the magnification effect of the enzyme-substrate reaction, fewer cells were needed for detection than with enrichment serology, thereby reducing the total analysis time. The ability to test 14 or more samples simultaneously on a 47-mm membrane filter would facilitate screening large numbers of samples. Pending the development of a pure H antisera pool for the common Salmonella serotypes free from 0 antibodies, the ELAT demonstrated potential as a Salmonella detection methodology.
Foods and feeds have traditionally been screened for Salmonella by the 5-day cultural procedure as recommended by the Association of Official Analytical Chemists (2) and the Bacteriological Analytical Manual (6). Accelerated Salmonella detection methodologies have been developed to reduce the analysis time. Of these, fluorescent-antibody techniques (11, 12, 16) and enrichment serology (4, 18) have been most popular. Fluorescent-antibody techniques are less reliable than cultural procedures, producing a high percentage of false-positive and false-negative results (7, 13). Results with enrichment serology, as described by Sperber and Deibel (18), agree more closely with results obtained by cultural procedures (13). Using the enrichment culture techniques of Sperber and Deibel (18), the indirect enzyme-labeled antibody technique (ELAT) described herein employs specific rabbit antiflagella antibody and peroxidase-labeled goat anti-rabbit antibody to detect Salmonella in food samples. Enzyme-labeled antibodies have been used to detect specific antigenic determinants in histo-
chemical and virological studies. Procedures to couple enzymes to antibodies, retaining both enzymatic and serological activity, have been described (3, 15). Nakane (14) used peroxidaselabeled antibody to detect six hormones of the anterior pituitary gland with light and electron microscopes. Horseradish peroxidase, alkaline phosphatase, and glucose oxidase were conjugated to virus antibody by Wicher and Avrameas (23) and used to detect adenovirus 12, simian virus 40, and rat K virus in infected cell cultures. Ubertini et al. (21) used peroxidasecoupled antibody to detect reovirus in tissue culture. These methods utilized the specificity of pure antibody coupled to enzyme to localize specific antigens. Upon application of substrate for enzyme, reaction product, which was detectable by microscopy, developed at the antigenic sites. This report differs from previous studies in that a membrane filter was the matrix for the technique and the results were read macroscopically.
I Approved by the Director of the Idaho Agriculture Experiment Station as Research Paper no. 7659. 2 Present address: Food Research Institute, University of Wisconsin, Madison, WI 53706.
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MATERIALS AND METHODS Organisms. S. typhimurium was obtained from the Idaho Department of Health and Welfare. The serotype was confirmed by typing with specific sera (Difco). The physiological reactions of the organisms were typical for S. typhimurium.
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Citrobacter freundii, Escherichia coli, Enterobacter aerogenes, Proteus vulgaris, Serratia marcescens, Pseudomonas fluorescens, an Erwinia species, and a Proteus species which was serologically and physiologically similar to a salmonella except for the urease reaction were obtained from the Department of Bacteriology and Biochemistry, University of Idaho. Spotting membranes. The ELAT employed cellulose-acetate membrane filters (Gelman Instrument Co.), 0.2- or 0.45-,um pore size, 25 or 47 mm in
diameter. Cellulose-nitrate membrane filters (Millipore Corp.), 0.45-,.um pore size, were also tested. A water-soaked membrane was placed over a Millipore filter holder base and covered with a water film; then a lucite template (Fig. 1) was placed over the membrane. Lucite templates bored with 7 and 14 wells were used on 25- and 47-mm membranes, respectively. Amounts of 0.1 ml of broth cultures or culture dilutions in phosphate-buffered saline (PBS), 0.01 M phosphate at pH 7.2, were dispensed in individual template wells, and the cells were spotted on membranes by applying suction. For sensitivity testing dilutions of S. typhimurium, M-broth (18) or brain heart infusion (BHI) cultures were diluted to contain approximately 107, 106, 105, 104, and 103 cells/0.1 ml. Cultures of organisms employed as specificity controls were diluted to contain approximately 107 cells/0.1 ml. Fixation. "4C-labeled S. typhimurium cells were used to establish conditions necessary to fix cells to the membrane filters. S. typhimurium was inoculated into 30 ml of filter-sterilized M-broth containing 60 ,ul of "4C-labeled L-amino acids (1 mCi/ml in 0.01 N HCl; CalAtomic), and the culture was incubated at 30°C for 15 h (1). Cells were collected by centrifugation at 12,000 x g for 30 min at 4°C, the supernatant culture fluid was discarded, and the pellet was gently suspended in 3 ml of PBS. After two PBS washes, cells were resuspended in 3 ml of PBS, and a 10 ,ul portion was spotted on individual (A) % I #-%
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5/8
/
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FIG.
1. Schematic
drawing of lucite template for
25-mm membrane filters. (A) Top view. (B) Well
dimensions; cross-sectional view.
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cellulose-acetate membrane filters cut to 10-mm diameters. Spotted membranes were air-dried. Fixation parameters tested were air drying and exposure to glutaraldehyde and formaldehyde. Spotted membranes were placed in baths containing aqueous concentrations of glutaraldehyde or formaldehyde, 0.005 to 1.0%, for 1 to 60 min and then airdried. To remove nonfixed cells, individual membranes were placed in 500-ml triple-baffled shake flasks containing 100 ml of PBS and were washed on a rotary shaker at 150 rpm for 10 min. To monitor fixation, washed membranes were transferred into scintillation vials containing cocktail to dissolve the membrane (17) and were counted for radioactivity with a liquid scintillation counter (model 3320, Packard Instrument Co.). Unwashed membranes spotted with "4C-labeled S. typhimurium were also counted. Buffers. All buffers employed were prepared by titrating equimolar solutions of acidic and basic components to the desired pH. Flagella purification. Flagella were purified according to Aleksic and Rohde (1). S. typhimurium was grown in swarm plates. Cells were suspended in buffer, sheared of flagella in an Omni Mixer (Ivan Sorvall, Inc.), and separated by differential centrifugation. The flagella were purified by diethylaminoethyl (DEAE)-cellulose chromatography. Fractions of the flagella peak were pooled, pelleted by ultracentrifugation at 106,000 x g for 60 min, and suspended in 0.02 M borate buffer, pH 8.4, to give a concentration of approximately 1 mg of protein/ml. Protein concentrations were estimated by absorbancy ratios at 260 and 280 nm. Tests for purity. Sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis was performed as outlined by Weber and Osborn (22), but the method was modified by using 0.05 M tris(hydroxymethyl)aminomethane (Tris)-acetate buffer, pH 8.1 (19). Samples of purified flagella were dissociated into monomeric flagellin units by mixing with four volumes of 0.005 M Tris-acetate buffer, pH 8.1, containing 20% sucrose, 4 M urea, 1% f3-mercaptoethanol, 1% SDS, and 0.005% bromophenol blue. After 2 to 4 h at 25°C, 10- to 20-,ul samples were layered on 10% polyacrylamide gels 10 cm in length. Electrophoresis was performed at 2 V/gel for 30 min and then 10 V/gel until the tracking dye reached the bottom of the gel. Rabbit immunization and H-antiserum preparation. DEAE-cellulose-purified flagella, 1 mg in 1 ml of 0.02 M borate buffer, were emulsified 1:1 with Freund complete adjuvant (Difco). Two male, albino, New Zealand rabbits received footpad injections of 0.5 ml/rabbit. A second 0.5-ml footpad injection of the same preparation was given 4 weeks later. During immunization, serum was obtained by bleeding from the marginal ear vein, and H-antibody titers were determined by tube agglutination as described by Edwards and Ewing (5). Blood was collected by cardiac puncture at 10- to 14-day intervals beginning 5 weeks after the first injection and continuing for the next 8 weeks. Harvested sera were stored at -60°C. The gamma globulin fraction (immunoglobulin G
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[IgG]) was separated from rabbit H antisera by gel filtration through a Sephadex G-200 (Pharmacia Fine Chemicals) column. The IgG peak was determined by using electrophoretically pure rabbit IgG (Schwarz/Mann) as a marker. IgG fractions were pooled, dialyzed at 4°C against distilled water, clarified by centrifugation, and lyophilized. Portions of this partially purified IgG pool were dissolved in PBS and used as the source of flagellar antibody in the ELAT. Commercial antiserum, polyvalent rabbit antiSalmonella flagella (Difco), diluted 1:25 in PBS was also tested in the ELAT. Goat anti-rabbit peroxidase conjugate preparation. Goat anti-rabbit IgG serum was kindly provided by R. A. Mead, Department of Biological Sciences, University of Idaho. The IgG fraction of whole goat serum was partially purified by precipitation with ammonium sulfate at 50% saturation (8). The precipitate was dissolved to original volume in distilled water, dialyzed against distilled water at 4°C, clarified by centrifugation, and lyophilized. Horseradish peroxidase, Per (type VI, Sigma Chemical Co.), was conjugated to salt-fractionated goat anti-rabbit IgG, GAR, by use of glutaraldehyde as described by Avrameas (3). After conjugation, the material, designated crude GAR-Per, was stored at -600C. To monitor conjugation and to remove uncoupled peroxidase and antibody from the conjugate, crude GAR-Per was fractionated by gel filtration through a Sephadex G-200 column. For reference, samples of salt-fractionated GAR and GAR containing uncoupled peroxidase were chromatographed through the column. Fractions corresponding to the GAR-Per conjugate were pooled, concentrated to the original sample volume with a collodion membrane apparatus (Brinkmann Instruments Inc.), and stored at -600C. Both GAR-Per preparations, crude and Sephadexpurified, were tested in the ELAT. Peroxidase assay. Peroxidase assays employed 3,3'-dimethoxybenzidine (Sigma) and H202 as substrates (24). Assays were performed at 460 nm with a recording spectrophotometer equipped with a 25°C bath (model 2400-2; Gilford Instrument Laboratories). Serological assay of conjugates. The titer of GAR-Per preparations was determined by latex particle agglutination. A procedure similar to that of Gordon and Lapa (9) was followed except that latex particles were used in place of charcoal particles. Latex particles (Difco) were sensitized with 0.125 mg of pure rabbit IgG/ml. Amounts of 100 ,ul of serial dilutions of GAR-Per were mixed with 5 ,u of sensitized particles on ring slides. After mixing, the slides were rotated at 100 rpm for 10 min at 20°C and observed for agglutination. ELAT. Spotted, fixed membranes were placed in a 50- or 60-mm petri dish containing 7 to 10 ml of lyophilized rabbit anti-H dissolved in PBS. Concentrations of 0.01 to 0.07 mg of anti-H/ml with contact times of 10 to 60 min and incubation at 25 to 37°C were tested. During incubation, anti-H was periodically repipetted over the membranes. The lowest
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antibody concentration applied for the shortest time that achieved maximal sensitivity was selected as the optimal concentration for the anti-H reaction. Unreacted antibody was removed by bathing the membranes in several changes of PBS followed by filtration with PBS. Membranes were then placed in a 7- to 10-ml bath of GAR-Per and incubated as above. GAR-Per concentrations ranging from undiluted to 1:100 in PBS were tested. The goat anti-rabbit reaction was optimized as above. After incubation, a similar washing procedure removed unreacted material from the membranes. S. typhimurium detection was accomplished by submerging membranes in substrate for 1 to 10 min. A localized brown reaction product developed with 0.03% 3,3'-diaminobenzidine (Sigma) in 0.05 M Trishydrochloride buffer, pH 7.6, containing 0.01% H202 (10). A localized blue reaction product developed with saturated aqueous benzidine (about 0.04%) containing 0.03% H202, followed by a 15-min stabilization in a bath of cold nitroprusside solution and a 95% ethanol wash (20). Food-sample preparation. Reconstituted nonfat dried milk (NFDM) was inoculated with S. typhimurium; the mixture was lyophilized and then ground to a fine powder. The inoculating powder contained 1.5 x 105 S. typhimurium cells/g (95% confidence limits: 0.3 x 105 to 4.4 x 105) as determined by a three-tube most probable number in BHI followed by selective plating on brilliant green agar and cultural analysis. Commercial pancake mix, NFDM, and trout feed were mixed with the inoculum in a Patterson twin shell blender (Patterson-Kelly Co., Inc.) to contain 50 S. typhimurium cells/g. Dilutions (wt/wt) were made in the blender to obtain levels of 10, 2, and 0.1 S. typhimurium cells/g of food. Food-sample testing. Enrichment serology, cultural analysis, and the ELAT using the enrichment technique of Sperber and Deibel (18) were performed concurrently. Duplicate 25-g samples of uninoculated and inoculated foods at each S. typhimurium level were preenriched for 18 h in lactose broth at 37°C, selectively enriched in selenite-cystine and tetrathionate broths for 24 h at 37°C, and inoculated into M-broth. M-broth was incubated at 37°C for 4 and 8 h for the ELAT and 8 h for serological detection by enrichment serology (18). For cultural analysis, selective plating, physiological testing, and serological detection followed selective enrichment (2).
RESULTS Fixation. When spotted, dried membranes were exposed to glutaraldehyde or formaldehyde solutions and dried again, increased numbers of cells remained on the membranes after a rotary-shaker wash. Exposure for 10 min to 0.05% glutaraldehyde and 0.01% formaldehyde fixed cells as effectively as higher concentrations and longer exposure times. The number of cells fixed decreased when membranes were exposed to 0.005% glutaraldehyde and formal-
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dehyde. Results from a representative fixation study employing air drying and 0.01 and 0.05% glutaraldehyde and formaldehyde are shown in Table 1. Greatest fixation, 90%, was obtained with a 10-min exposure to 0.05% glutaraldehyde. Air drying alone fixed 45% of the spotted S. typhimurium cells. These fixation parameters were tested in two additional trials and the results were averaged. Means obtained with 0.05% glutaraldehyde and air drying were 83% (range, 78 to 90) and 43% (range, 32 to 52) fixation, respectively. For comparison in the ELAT, identically spotted membranes were fixed by air drying (minimal fixation) and exposure to 0.05% glutaraldehyde for 10 min (maximal fixation). Flagella purification and anti-S. typhimurium H antibody preparation. When samples
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of partially purified flagella obtained by differential centrifugation were chromatographed on DEAE-cellulose (Fig. 2), flagella (second peak of absorbance) were readily resolved from other absorbing materials. Fractions containing purified flagella from several experiments were pooled. The pooled fractions were concentrated by ultracentrifugation, and portions were tested for electrophoretic homogeneity. Since the purified flagella pool yielded a single protein band by SDS-polyacrylamide gel electrophoresis (Fig. 3), the preparation was judged to be of high purity and a suitable antigen for rabbit immunization.
TABLE 1. Fixation of 14C-labeled S. typhimurium to cellulose-acetate membrane filters" Counts/2 minb Cells (%) ingremain-
Treatment
No wash control .......... 1,993 Air driedc ................ 889 Glutaraldehyde, 0.05%d ... 1,790
100 45 90 ...0 1,506 76 Glutaraldehyde, 00%d 72 Formaldehyde, 0.05%d .... 1,425 74 Formaldehyde, 0.01%d .... 1,484 a Membranes spotted with 10 ,ul of '4C-labeled S. typhimurium suspension. b Average scintillation count of duplicate samples. ' Rotary shake flask wash (10 min, 150 rpm) applied after fixation. d Exposure to fixative for 10 min, followed by 10min, 150-rpm rotary shake flask wash. 0.7
C 0 04
0.3[ b
a
0.I
I "
20
40
60
80
FRACTION NO. (3 ml/FRACTION)
FIG. 2. DEAE-cellulose chromatography elution profile of flagella purified by differential centrifugation. Column (1.5 by 20 cm), equilibrated with 0.02 M borate buffer, pH 8.4; sample, 10 ml containing 14 mg of protein; flow rate, 1.5 ml/min; flagella peak, fractions 82 through 90, saved. (a) Elution with 0.02 M borate buffer containing 0.2 M NaC1, pH 8.4; (b) elution with 0.02 M borate buffer containing 0.4 M NaCl, pH 8.4.
FIG. 3. SDS-polyacrylamide gel electrophoresis of DEAE-cellulose-purified flagella. Sample, 20 ug of protein denatured in buffer containing ,3mercaptoethanol and SDS. Symbols: +, anode; -, cathode.
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(Fig. 4C) would have relatively more protein eluting with the column void volume than would equivalent samples containing uncoupled antibody and peroxidase (Fig. 4B). The Sephadex-purified GAR-Per (Fig. 4C) contained 16% of the total enzymatic activity and 25% of the total serological activity of the crude GAR-Per sample. ELAT methodology. Optimal results were achieved by immersing membranes spotted with S. typhimurium in a solution of 1 mg of rabbit anti-H IgG dissolved in 60 ml of PBS (half the concentration required for cellular tube agglutination) for 30 min. Incubation at 25 or 37°C produced equal results. After a wash, immersing the anti-H-treated membranes in a 1:50 PBS dilution of crude GAR-Per (0.03 mg of goat IgG/ml) for 15 min gave optimal results. Incubation at 25 or 370C again yielded identical results. Since enzymatic and serological activity of crude GAR-Per was lost during gel filtration and concentration, a higher concentration of Sephadex-purified conjugate was required to achieve comparable sensitivity. Since uncoupled peroxidase, uncoupled 4.01 antibody, and unreacted antibody-enzyme conA jugate in crude GAR-Per were completely washed from the cellulose-acetate membranes 2.0 producing essentially no background reaction product upon exposure to substrates, crude GAR-Per was routinely used in the ELAT. Comparable S. typhimurium sensitivity was achieved with either 3,3'-diaminobenzidine or benzidine as substrate. Representative results Ec are shown in Fig. 5. With both substrates, spots containing 104 or more S. typhimurium cells in were consistently positive by macroscopic examination. Frequently, spots containing 103 S. typhimurium cells gave positive results. The lack of a positive reaction for the spot containing 104 S. typhimurium cells on membrane III (Fig. 5) was not due to a flaw in the ELAT, but to fading of the benzidine reaction product before the photograph was taken. Mixed cultures did not diminish sensitivity of the technique. Spots containing 107, 106, and 105 S. typhimurium cells plus 107 E. coli or Proteus sp. cells FRACTION NO. (4.5 ml/FRACTION) gave essentially identical results when comFIG. 4. Gel-filtration elution profiles of uncoupled pared with spots containing the same quantiGAR, uncoupled GAR plus uncoupled Per, and ties of S. typhimurium cells alone. Since spotcrude GAR-Per conjugate. Column, Sephadex Gted membranes fixed by air drying or exposure 200, 2.5 by 85 cm; buffer, pH 7.2 PBS; flow rate, 32 to glutaraldehyde behaved the same when ml/h; void volume, fraction 38. (A) Elution profile of tested as described above, air drying was the uncoupled goat antirabbit IgG (GAR). Sample, 3 ml preferred fixation. containing 6 mg; IgG peak, fractions 45 throught 58. The ELAT was specific for S. typhimurium. (B) Elution profile of uncoupled GAR plus uncoupled No false-positive reactions were experienced Per. Sample, 3 ml containing 5 mg of GAR and 6 mg when cultures of C. freundii, E. coli, E. aeroof Per; peroxidase peak, fractions 60 through 77. (C) Elution profile of crude GAR-Per conjugate. Gar-Per genes, P. vulgaris, S. marcescens, P. fluoresconjugate, fractions 38 through 43, saved. cens, an Erwinia sp., and a Proteus sp. were
Within 5 weeks after the first injection, flagella-immunized rabbits responded with serum agglutination titers of 1:8,192 which were maintained for several weeks. During this period sera were collected. The IgG fractions were isolated by gel filtration, pooled, and lyophilized. When the partially purified IgG powder was dissolved in PBS (1 mg/ml), the solution had an agglutination titer of 1:32. GAR-Per. Conjugation of peroxidase (Per) to the IgG fraction of goat anti-rabbit IgG serum (GAR) was demonstrated by comparing the gelfiltration elution profile of crude GAR-Per conjugate (Fig. 4C) with the elution profiles of uncoupled GAR alone (Fig. 4A) and uncoupled GAR plus uncoupled Per (Fig. 4B). Molecular conjugates containing both antibody and enzyme would have a molecular weight of 190,000 or greater (150,000 for IgG plus 40,000 for peroxidase), a molecular size at or near the exclusion limit of Sephadex G-200. Therefore, samples containing antibody-peroxidase conjugates
IA 1-
0
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FIG. 5. Representative ELAT results with 25-mm cellulose-acetate membrane filters. Center membrane shows the template well pattern. Membranes I and II were developed in 3,3'-diaminobenzidine (-DAB-). Membranes III and IV were developed in benzidine (-B-). Membranes I and III were spotted as follows: spots 1-5 contained 107, 106, 105, 104, and 103 S. typhimurium cells, respectively; spot 6 contained 107 Proteus sp. cells; spot 7 contained 107 E. coli cells. Membranes II and IV were spotted as follows: spot 1 contained 107 S. typhimurium cells; spots 2-4 contained 107, 106, and 105 S. typhimurium cells, respectively, each mixed with 107 Proteus sp. cells; spots 5-7 contained 107, 106, and 105 S. typhimurium cells, respectively, each mixed with 107 E. coli cells.
tested. Specificity was lost when Difco poly-H antiserum was substituted for anti-S. typhimurium H antibody; C. freundii, E. coli, E. aerogenes, P. fluorescens, the Erwinia sp., and the Proteus sp. produced weak false-positive reactions. This may reflect the presence of nonspecific 0 antibody present in the Difco H-antiserum (7). Millipore (cellulose-nitrate) membranes were unsuitable for this methodology. Unreacted reagents could not be completely washed from membranes before exposure to substrate. This resulted in a background enzymatic product on membranes which masked the positive enzymatic product for S. typhimurium. When Gelman (cellulose-acetate) membranes were used, a blue reaction product with benzidine as substrate was preferred. Occasionally, insoluble material in enrichment broths, especially Mbroth inoculated with selenite-cystine broth, left a brown deposit on membranes which was confused with the brown enzymatic product which developed with 3,3'-diaminobenzidine. Nonspecific, false positives were not encoun-
tered when benzidine was the developing substrate. Tests in food products. Commercial pancake mix, NFDM, and commercial trout feed not inoculated with S. typhimurium were Salmonella negative by enrichment serology, cultural procedures, and the ELAT. At levels of 50, 10, and 2 S. typhimurium cells/g, each of the methods detected the presence of salmonellae in all food samples tested. In a comparison between enrichment serology and the ELAT on samples containing one S. typhimurium cell/10 g (95% confidence limits: 0.2 to 2.9), duplicate samples of NFDM were positive and pancake mix and fish feed were negative by both methods. For the ELAT, best results were obtained by spotting M-broth cultures showing the first sign of turbidity. In this study M-broth turbidity developed after approximately 4 h of incubation. This cell density was optimal for spotting membranes, and the spots contained sufficient numbers of S. typhimurium for detection. Because of the high cell density, 8-h M-broth cultures had to be diluted, usually 1:4, before spotting.
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DISCUSSION For a new Salmonella detection methodology to be acceptable, it must offer some advantage or improvement over existing methodologies. The ELAT appears to satisfy these criteria. The ELAT uses a filter membrane as a matrix for detection. A 47-mm membrane can be spotted with 14 or more broth enrichment samples, facilitating multisample analyses. The procedure requires little technical expertise or equipment and is relatively inexpensive to perform. In this study 100 mg of peroxidase coupled to antibody was sufficient to test over 10,000 Salmonella spots. Detection occurred through the amplification effect of the enzyme-substrate reaction, producing a macroscopically visible deposit of insoluble product on membranes spotted with broth cultures containing S. typhimurium. In contrast to the 5 x 107 salmonellae/ml needed for detection by enrichment serology or fluorescent-antibody techniques (18), the ELAT was sensitive to 105, and to a lesser extent 104, S. typhimurium cells/ml. When Sperber and Deibel's enrichment broths were used, the ELAT performed on M-broth cultures showing the first sign of turbidity (about 4 h) agreed with the results obtained by enrichment serology and cultural procedures on three food products containing low levels of S. typhimurium. The total analysis time was 48 h: 18 h of preenrichment, 24 h of selective enrichment, 4 h in Mbroth, a 2 h for the ELAT. The procedure demonstrated Salmonella specificity when pure H antisera free from 0 antibodies were used. False-positive results ocurred with Difco poly H antisera, probably as a result of the presence of nonspecific 0 antibodies. This may represent a limiting factor in the ELAT since a pool of pure H antisera for common Salmonella serotypes appears to be necessary before the method would be of value in the routine surveillance of Salmonella in foods. Both cellulose-acetate membranes and a benzidine substrate are recommended for the methodology. Cellulose-nitrate membranes produced unsatisfactory results because residual reactants which could not be completely removed resulted in background enzymatic product. False-positive results encountered with 3,3'-diaminobenzidine were eliminated by using benzidine as substrate. The resulting blue reaction product could only be interpreted as an enzymatic reaction product and could not be confused with nonenzymatic brown deposits from enrichment media.
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A chemical fixation step to prevent spotted cells from washing off membranes was not needed. Membranes which were air-dried after spotting produced results similar to membranes fixed in glutaraldehyde. This can be attributed to the mild washes employed in the methodology and the fixation inherent to air drying. The washes used were sufficient to remove unreacted reagents from membranes; thus, purification of crude GAR-Per was not necessary. This study demonstrated that enzyme-labeled antibodies can be used as a sensitive and specific methodology for macroscopic detection of Salmonella in mixed enrichment cultures. Further work using a polyvalent H antisera on many food samples is needed. A similar enzyme-labeled antibody method could be adapted for clinical analyses or in other systems where specific antigenic determinants are available for detection. ACKNOWLEDGMENTS This work was supported by the University of Idaho Agricultural Research Experiment Station under project 708-R670. LITERATURE CITED 1. Aleksic, S., and R. Rohde. 1972. The separation and
purification of Salmonella-Arizona H-antigens by DEAE-cellulose chromatography for the preparation and purification of diagnostic H-antisera with high titers and free of 0-antibodies. Ann. Inst. Pasteur Paris 123:363-370. 2. Association of Official Analytical Chemists. 1970. Official methods of analysis, 11th ed. Association of Official Analytical Chemists, Washington, D.C. 3. Avrameas, S. 1969. Coupling of enzymes to proteins with glutaraldehyde. Immunochemistry 6:43-52. 4. Barkata, J. A. 1968. Screening of feed components for Salmonella with polyvalent H agglutination. Appl. Microbiol. 16:1872-1874. 5. Edwards, D. R., and W. H. Ewing. 1972. Identification of Enterobacteriaceae, 3rd ed. Burgess Publishing Co., Minneapolis, Minn. 6. Elliot, R. P. (ed.). 1966. Bacteriological analytical manual. U.S. Food and Drug Administration, Washington, D.C. 7. Goepfert, J. M., and R. Hicks. 1969. Immunofluorescent staining of Salmonella species with flagella sera. Appl. Microbiol. 18:612-617. 8. Goldman, M. 1968. Fluorescent antibody methods, p. 94. Academic Press Inc., New York. 9. Gordon, M. A., and E. Lapa. 1971. Charcoal particle agglutination test for detection of antibody to Cryptococcus neoformas. Am. J. Clin. Pathol. 56:354-359. 10. Grahm, R. C., Jr., and M. J. Karnovsky. 1966. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural chemistry by a new technique. J. Histochem. Cytochem. 14:291-302. 11. Insalata, N. F., C. W. Mahnke, and W. G. Dunlap. 1974. Rapid, direct fluorescent antibody method for the detection of Salmonella in foods and feeds. Appl. Microbiol. 24:645-649. 12. Insalata, N. F., S. Schulte, and J. Berman. 1967. Immunofluorescent technique for detection of Salmo-
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nella in various foods. Appl. Microbiol. 15:1145-1149. 13. Mohr, H. K., H. C. Trenk, and M. Yeterian. 1970. Comparison of fluorescent antibodv methods and enrichment serology for the detection of Salmonella. Appl. Microbiol. 27:324-328. 14. Nakane, P. K. 1970. Classification of anterior pituitary cell types with immunoenzyme histochemistry. J. Histochem. Cytochem. 18:9-20. 15. Nakane, P. K., and G. B. Pierce, Jr. 1967. Enzymelabeled antibodies for the light and electron microscope localization of tissue antigens. J. Cell Biol. 33:307-317. 16. Reamer, R. H., and R. Hargrove. 1972. Twenty-fourhour immunofluorescence technique for the detection of Salmonella in nonfat dried milk. Appl. Microbiol. 23:78-81. 17. Rhodes, B. 1965. Liquid scintillation counting of radioiodine. Anal. Chem. 37:995. 18. Sperber, W. H., and R. H. Deibel. 1969. Accelerated procedure for Salmonella detection in dried foods and feeds involving only broth cultures and serological reactions. Appl. Microbiol. 17:553-539.
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Strafford, W. F., and D. A. Yphantis. 1972. Existence and inhibition of hydrolytic enzymes attacking paramyosin in myofibrillar extracts of Mercenaria mercenaria. Biochem. Biophys. Res. Commun. 49:848-845. 20. Straus, W. 1964. Factors affecting the cytochemical reaction of peroxidase with benzidine and the stability of the blue reaction product. J. Histochem. Cytochem. 12:462-468. 21. Ubertini, T., B. Wilkie, and F. Noronha. 1971. Use of 19.
horseradish peroxidase-labeled antibody for light and electron microscope localization of reovirus antigen. Appl. Microbiol. 21:534-538. 22. Weber, K., and M. Osborn. 1969. The reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406-4412. 23. Wicker, R., and S. Avrameas. 1969. Localization of virus antigens by enzyme-labeled antibodies. J. Gen. Virol. 4:465-468. 24. Worthington Biochemical Corp. 1972. Worthington enzyme manual. Worthington Biochemical Corp., Freehold, N.J.
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Vol. 33, No. 4 Printed in U.S.A.
Use of Enzyme-Labeled Antibodies to Detect Salmonella in Foods1 E. P. KRYSINSKI2 AND R. C. HEIMSCH* Department of Bacteriology and Biochemistry, University ofIdaho, Moscow, Idaho 83843
Received for publication 29 November 1976
An indirect enzyme-labeled antibody technique (ELAT), in which Salmonella typhimurium was used as a model, was developed as a method to detect Salmonella in food samples. A cellulose-acetate membrane filter, the matrix for detection, was placed on a membrane-filter base and overlaid with a multiwelled lucite template. Mixed broth enrichment cultures were dispensed in the template wells, and cells were spotted onto the membrane via suction. After fixation, the membranes were immersed in rabbit anti-S. typhimurium flagella antibody, washed, immersed in goat anti-rabbit antibody conjugated to peroxidase, and washed. Exposure of membranes to the substrates 3,3'-diaminobenzidine or benzidine resulted in development of brown or blue macroscopic reaction products, respectively, on spots containing S. typhimurium. ELAT results agreed with those of enrichment serology and cultural procedures on three food products containing known levels of S. typhimurium. Because of the magnification effect of the enzyme-substrate reaction, fewer cells were needed for detection than with enrichment serology, thereby reducing the total analysis time. The ability to test 14 or more samples simultaneously on a 47-mm membrane filter would facilitate screening large numbers of samples. Pending the development of a pure H antisera pool for the common Salmonella serotypes free from 0 antibodies, the ELAT demonstrated potential as a Salmonella detection methodology.
Foods and feeds have traditionally been screened for Salmonella by the 5-day cultural procedure as recommended by the Association of Official Analytical Chemists (2) and the Bacteriological Analytical Manual (6). Accelerated Salmonella detection methodologies have been developed to reduce the analysis time. Of these, fluorescent-antibody techniques (11, 12, 16) and enrichment serology (4, 18) have been most popular. Fluorescent-antibody techniques are less reliable than cultural procedures, producing a high percentage of false-positive and false-negative results (7, 13). Results with enrichment serology, as described by Sperber and Deibel (18), agree more closely with results obtained by cultural procedures (13). Using the enrichment culture techniques of Sperber and Deibel (18), the indirect enzyme-labeled antibody technique (ELAT) described herein employs specific rabbit antiflagella antibody and peroxidase-labeled goat anti-rabbit antibody to detect Salmonella in food samples. Enzyme-labeled antibodies have been used to detect specific antigenic determinants in histo-
chemical and virological studies. Procedures to couple enzymes to antibodies, retaining both enzymatic and serological activity, have been described (3, 15). Nakane (14) used peroxidaselabeled antibody to detect six hormones of the anterior pituitary gland with light and electron microscopes. Horseradish peroxidase, alkaline phosphatase, and glucose oxidase were conjugated to virus antibody by Wicher and Avrameas (23) and used to detect adenovirus 12, simian virus 40, and rat K virus in infected cell cultures. Ubertini et al. (21) used peroxidasecoupled antibody to detect reovirus in tissue culture. These methods utilized the specificity of pure antibody coupled to enzyme to localize specific antigens. Upon application of substrate for enzyme, reaction product, which was detectable by microscopy, developed at the antigenic sites. This report differs from previous studies in that a membrane filter was the matrix for the technique and the results were read macroscopically.
I Approved by the Director of the Idaho Agriculture Experiment Station as Research Paper no. 7659. 2 Present address: Food Research Institute, University of Wisconsin, Madison, WI 53706.
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MATERIALS AND METHODS Organisms. S. typhimurium was obtained from the Idaho Department of Health and Welfare. The serotype was confirmed by typing with specific sera (Difco). The physiological reactions of the organisms were typical for S. typhimurium.
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Citrobacter freundii, Escherichia coli, Enterobacter aerogenes, Proteus vulgaris, Serratia marcescens, Pseudomonas fluorescens, an Erwinia species, and a Proteus species which was serologically and physiologically similar to a salmonella except for the urease reaction were obtained from the Department of Bacteriology and Biochemistry, University of Idaho. Spotting membranes. The ELAT employed cellulose-acetate membrane filters (Gelman Instrument Co.), 0.2- or 0.45-,um pore size, 25 or 47 mm in
diameter. Cellulose-nitrate membrane filters (Millipore Corp.), 0.45-,.um pore size, were also tested. A water-soaked membrane was placed over a Millipore filter holder base and covered with a water film; then a lucite template (Fig. 1) was placed over the membrane. Lucite templates bored with 7 and 14 wells were used on 25- and 47-mm membranes, respectively. Amounts of 0.1 ml of broth cultures or culture dilutions in phosphate-buffered saline (PBS), 0.01 M phosphate at pH 7.2, were dispensed in individual template wells, and the cells were spotted on membranes by applying suction. For sensitivity testing dilutions of S. typhimurium, M-broth (18) or brain heart infusion (BHI) cultures were diluted to contain approximately 107, 106, 105, 104, and 103 cells/0.1 ml. Cultures of organisms employed as specificity controls were diluted to contain approximately 107 cells/0.1 ml. Fixation. "4C-labeled S. typhimurium cells were used to establish conditions necessary to fix cells to the membrane filters. S. typhimurium was inoculated into 30 ml of filter-sterilized M-broth containing 60 ,ul of "4C-labeled L-amino acids (1 mCi/ml in 0.01 N HCl; CalAtomic), and the culture was incubated at 30°C for 15 h (1). Cells were collected by centrifugation at 12,000 x g for 30 min at 4°C, the supernatant culture fluid was discarded, and the pellet was gently suspended in 3 ml of PBS. After two PBS washes, cells were resuspended in 3 ml of PBS, and a 10 ,ul portion was spotted on individual (A) % I #-%
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1. Schematic
drawing of lucite template for
25-mm membrane filters. (A) Top view. (B) Well
dimensions; cross-sectional view.
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cellulose-acetate membrane filters cut to 10-mm diameters. Spotted membranes were air-dried. Fixation parameters tested were air drying and exposure to glutaraldehyde and formaldehyde. Spotted membranes were placed in baths containing aqueous concentrations of glutaraldehyde or formaldehyde, 0.005 to 1.0%, for 1 to 60 min and then airdried. To remove nonfixed cells, individual membranes were placed in 500-ml triple-baffled shake flasks containing 100 ml of PBS and were washed on a rotary shaker at 150 rpm for 10 min. To monitor fixation, washed membranes were transferred into scintillation vials containing cocktail to dissolve the membrane (17) and were counted for radioactivity with a liquid scintillation counter (model 3320, Packard Instrument Co.). Unwashed membranes spotted with "4C-labeled S. typhimurium were also counted. Buffers. All buffers employed were prepared by titrating equimolar solutions of acidic and basic components to the desired pH. Flagella purification. Flagella were purified according to Aleksic and Rohde (1). S. typhimurium was grown in swarm plates. Cells were suspended in buffer, sheared of flagella in an Omni Mixer (Ivan Sorvall, Inc.), and separated by differential centrifugation. The flagella were purified by diethylaminoethyl (DEAE)-cellulose chromatography. Fractions of the flagella peak were pooled, pelleted by ultracentrifugation at 106,000 x g for 60 min, and suspended in 0.02 M borate buffer, pH 8.4, to give a concentration of approximately 1 mg of protein/ml. Protein concentrations were estimated by absorbancy ratios at 260 and 280 nm. Tests for purity. Sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis was performed as outlined by Weber and Osborn (22), but the method was modified by using 0.05 M tris(hydroxymethyl)aminomethane (Tris)-acetate buffer, pH 8.1 (19). Samples of purified flagella were dissociated into monomeric flagellin units by mixing with four volumes of 0.005 M Tris-acetate buffer, pH 8.1, containing 20% sucrose, 4 M urea, 1% f3-mercaptoethanol, 1% SDS, and 0.005% bromophenol blue. After 2 to 4 h at 25°C, 10- to 20-,ul samples were layered on 10% polyacrylamide gels 10 cm in length. Electrophoresis was performed at 2 V/gel for 30 min and then 10 V/gel until the tracking dye reached the bottom of the gel. Rabbit immunization and H-antiserum preparation. DEAE-cellulose-purified flagella, 1 mg in 1 ml of 0.02 M borate buffer, were emulsified 1:1 with Freund complete adjuvant (Difco). Two male, albino, New Zealand rabbits received footpad injections of 0.5 ml/rabbit. A second 0.5-ml footpad injection of the same preparation was given 4 weeks later. During immunization, serum was obtained by bleeding from the marginal ear vein, and H-antibody titers were determined by tube agglutination as described by Edwards and Ewing (5). Blood was collected by cardiac puncture at 10- to 14-day intervals beginning 5 weeks after the first injection and continuing for the next 8 weeks. Harvested sera were stored at -60°C. The gamma globulin fraction (immunoglobulin G
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[IgG]) was separated from rabbit H antisera by gel filtration through a Sephadex G-200 (Pharmacia Fine Chemicals) column. The IgG peak was determined by using electrophoretically pure rabbit IgG (Schwarz/Mann) as a marker. IgG fractions were pooled, dialyzed at 4°C against distilled water, clarified by centrifugation, and lyophilized. Portions of this partially purified IgG pool were dissolved in PBS and used as the source of flagellar antibody in the ELAT. Commercial antiserum, polyvalent rabbit antiSalmonella flagella (Difco), diluted 1:25 in PBS was also tested in the ELAT. Goat anti-rabbit peroxidase conjugate preparation. Goat anti-rabbit IgG serum was kindly provided by R. A. Mead, Department of Biological Sciences, University of Idaho. The IgG fraction of whole goat serum was partially purified by precipitation with ammonium sulfate at 50% saturation (8). The precipitate was dissolved to original volume in distilled water, dialyzed against distilled water at 4°C, clarified by centrifugation, and lyophilized. Horseradish peroxidase, Per (type VI, Sigma Chemical Co.), was conjugated to salt-fractionated goat anti-rabbit IgG, GAR, by use of glutaraldehyde as described by Avrameas (3). After conjugation, the material, designated crude GAR-Per, was stored at -600C. To monitor conjugation and to remove uncoupled peroxidase and antibody from the conjugate, crude GAR-Per was fractionated by gel filtration through a Sephadex G-200 column. For reference, samples of salt-fractionated GAR and GAR containing uncoupled peroxidase were chromatographed through the column. Fractions corresponding to the GAR-Per conjugate were pooled, concentrated to the original sample volume with a collodion membrane apparatus (Brinkmann Instruments Inc.), and stored at -600C. Both GAR-Per preparations, crude and Sephadexpurified, were tested in the ELAT. Peroxidase assay. Peroxidase assays employed 3,3'-dimethoxybenzidine (Sigma) and H202 as substrates (24). Assays were performed at 460 nm with a recording spectrophotometer equipped with a 25°C bath (model 2400-2; Gilford Instrument Laboratories). Serological assay of conjugates. The titer of GAR-Per preparations was determined by latex particle agglutination. A procedure similar to that of Gordon and Lapa (9) was followed except that latex particles were used in place of charcoal particles. Latex particles (Difco) were sensitized with 0.125 mg of pure rabbit IgG/ml. Amounts of 100 ,ul of serial dilutions of GAR-Per were mixed with 5 ,u of sensitized particles on ring slides. After mixing, the slides were rotated at 100 rpm for 10 min at 20°C and observed for agglutination. ELAT. Spotted, fixed membranes were placed in a 50- or 60-mm petri dish containing 7 to 10 ml of lyophilized rabbit anti-H dissolved in PBS. Concentrations of 0.01 to 0.07 mg of anti-H/ml with contact times of 10 to 60 min and incubation at 25 to 37°C were tested. During incubation, anti-H was periodically repipetted over the membranes. The lowest
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antibody concentration applied for the shortest time that achieved maximal sensitivity was selected as the optimal concentration for the anti-H reaction. Unreacted antibody was removed by bathing the membranes in several changes of PBS followed by filtration with PBS. Membranes were then placed in a 7- to 10-ml bath of GAR-Per and incubated as above. GAR-Per concentrations ranging from undiluted to 1:100 in PBS were tested. The goat anti-rabbit reaction was optimized as above. After incubation, a similar washing procedure removed unreacted material from the membranes. S. typhimurium detection was accomplished by submerging membranes in substrate for 1 to 10 min. A localized brown reaction product developed with 0.03% 3,3'-diaminobenzidine (Sigma) in 0.05 M Trishydrochloride buffer, pH 7.6, containing 0.01% H202 (10). A localized blue reaction product developed with saturated aqueous benzidine (about 0.04%) containing 0.03% H202, followed by a 15-min stabilization in a bath of cold nitroprusside solution and a 95% ethanol wash (20). Food-sample preparation. Reconstituted nonfat dried milk (NFDM) was inoculated with S. typhimurium; the mixture was lyophilized and then ground to a fine powder. The inoculating powder contained 1.5 x 105 S. typhimurium cells/g (95% confidence limits: 0.3 x 105 to 4.4 x 105) as determined by a three-tube most probable number in BHI followed by selective plating on brilliant green agar and cultural analysis. Commercial pancake mix, NFDM, and trout feed were mixed with the inoculum in a Patterson twin shell blender (Patterson-Kelly Co., Inc.) to contain 50 S. typhimurium cells/g. Dilutions (wt/wt) were made in the blender to obtain levels of 10, 2, and 0.1 S. typhimurium cells/g of food. Food-sample testing. Enrichment serology, cultural analysis, and the ELAT using the enrichment technique of Sperber and Deibel (18) were performed concurrently. Duplicate 25-g samples of uninoculated and inoculated foods at each S. typhimurium level were preenriched for 18 h in lactose broth at 37°C, selectively enriched in selenite-cystine and tetrathionate broths for 24 h at 37°C, and inoculated into M-broth. M-broth was incubated at 37°C for 4 and 8 h for the ELAT and 8 h for serological detection by enrichment serology (18). For cultural analysis, selective plating, physiological testing, and serological detection followed selective enrichment (2).
RESULTS Fixation. When spotted, dried membranes were exposed to glutaraldehyde or formaldehyde solutions and dried again, increased numbers of cells remained on the membranes after a rotary-shaker wash. Exposure for 10 min to 0.05% glutaraldehyde and 0.01% formaldehyde fixed cells as effectively as higher concentrations and longer exposure times. The number of cells fixed decreased when membranes were exposed to 0.005% glutaraldehyde and formal-
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dehyde. Results from a representative fixation study employing air drying and 0.01 and 0.05% glutaraldehyde and formaldehyde are shown in Table 1. Greatest fixation, 90%, was obtained with a 10-min exposure to 0.05% glutaraldehyde. Air drying alone fixed 45% of the spotted S. typhimurium cells. These fixation parameters were tested in two additional trials and the results were averaged. Means obtained with 0.05% glutaraldehyde and air drying were 83% (range, 78 to 90) and 43% (range, 32 to 52) fixation, respectively. For comparison in the ELAT, identically spotted membranes were fixed by air drying (minimal fixation) and exposure to 0.05% glutaraldehyde for 10 min (maximal fixation). Flagella purification and anti-S. typhimurium H antibody preparation. When samples
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of partially purified flagella obtained by differential centrifugation were chromatographed on DEAE-cellulose (Fig. 2), flagella (second peak of absorbance) were readily resolved from other absorbing materials. Fractions containing purified flagella from several experiments were pooled. The pooled fractions were concentrated by ultracentrifugation, and portions were tested for electrophoretic homogeneity. Since the purified flagella pool yielded a single protein band by SDS-polyacrylamide gel electrophoresis (Fig. 3), the preparation was judged to be of high purity and a suitable antigen for rabbit immunization.
TABLE 1. Fixation of 14C-labeled S. typhimurium to cellulose-acetate membrane filters" Counts/2 minb Cells (%) ingremain-
Treatment
No wash control .......... 1,993 Air driedc ................ 889 Glutaraldehyde, 0.05%d ... 1,790
100 45 90 ...0 1,506 76 Glutaraldehyde, 00%d 72 Formaldehyde, 0.05%d .... 1,425 74 Formaldehyde, 0.01%d .... 1,484 a Membranes spotted with 10 ,ul of '4C-labeled S. typhimurium suspension. b Average scintillation count of duplicate samples. ' Rotary shake flask wash (10 min, 150 rpm) applied after fixation. d Exposure to fixative for 10 min, followed by 10min, 150-rpm rotary shake flask wash. 0.7
C 0 04
0.3[ b
a
0.I
I "
20
40
60
80
FRACTION NO. (3 ml/FRACTION)
FIG. 2. DEAE-cellulose chromatography elution profile of flagella purified by differential centrifugation. Column (1.5 by 20 cm), equilibrated with 0.02 M borate buffer, pH 8.4; sample, 10 ml containing 14 mg of protein; flow rate, 1.5 ml/min; flagella peak, fractions 82 through 90, saved. (a) Elution with 0.02 M borate buffer containing 0.2 M NaC1, pH 8.4; (b) elution with 0.02 M borate buffer containing 0.4 M NaCl, pH 8.4.
FIG. 3. SDS-polyacrylamide gel electrophoresis of DEAE-cellulose-purified flagella. Sample, 20 ug of protein denatured in buffer containing ,3mercaptoethanol and SDS. Symbols: +, anode; -, cathode.
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(Fig. 4C) would have relatively more protein eluting with the column void volume than would equivalent samples containing uncoupled antibody and peroxidase (Fig. 4B). The Sephadex-purified GAR-Per (Fig. 4C) contained 16% of the total enzymatic activity and 25% of the total serological activity of the crude GAR-Per sample. ELAT methodology. Optimal results were achieved by immersing membranes spotted with S. typhimurium in a solution of 1 mg of rabbit anti-H IgG dissolved in 60 ml of PBS (half the concentration required for cellular tube agglutination) for 30 min. Incubation at 25 or 37°C produced equal results. After a wash, immersing the anti-H-treated membranes in a 1:50 PBS dilution of crude GAR-Per (0.03 mg of goat IgG/ml) for 15 min gave optimal results. Incubation at 25 or 370C again yielded identical results. Since enzymatic and serological activity of crude GAR-Per was lost during gel filtration and concentration, a higher concentration of Sephadex-purified conjugate was required to achieve comparable sensitivity. Since uncoupled peroxidase, uncoupled 4.01 antibody, and unreacted antibody-enzyme conA jugate in crude GAR-Per were completely washed from the cellulose-acetate membranes 2.0 producing essentially no background reaction product upon exposure to substrates, crude GAR-Per was routinely used in the ELAT. Comparable S. typhimurium sensitivity was achieved with either 3,3'-diaminobenzidine or benzidine as substrate. Representative results Ec are shown in Fig. 5. With both substrates, spots containing 104 or more S. typhimurium cells in were consistently positive by macroscopic examination. Frequently, spots containing 103 S. typhimurium cells gave positive results. The lack of a positive reaction for the spot containing 104 S. typhimurium cells on membrane III (Fig. 5) was not due to a flaw in the ELAT, but to fading of the benzidine reaction product before the photograph was taken. Mixed cultures did not diminish sensitivity of the technique. Spots containing 107, 106, and 105 S. typhimurium cells plus 107 E. coli or Proteus sp. cells FRACTION NO. (4.5 ml/FRACTION) gave essentially identical results when comFIG. 4. Gel-filtration elution profiles of uncoupled pared with spots containing the same quantiGAR, uncoupled GAR plus uncoupled Per, and ties of S. typhimurium cells alone. Since spotcrude GAR-Per conjugate. Column, Sephadex Gted membranes fixed by air drying or exposure 200, 2.5 by 85 cm; buffer, pH 7.2 PBS; flow rate, 32 to glutaraldehyde behaved the same when ml/h; void volume, fraction 38. (A) Elution profile of tested as described above, air drying was the uncoupled goat antirabbit IgG (GAR). Sample, 3 ml preferred fixation. containing 6 mg; IgG peak, fractions 45 throught 58. The ELAT was specific for S. typhimurium. (B) Elution profile of uncoupled GAR plus uncoupled No false-positive reactions were experienced Per. Sample, 3 ml containing 5 mg of GAR and 6 mg when cultures of C. freundii, E. coli, E. aeroof Per; peroxidase peak, fractions 60 through 77. (C) Elution profile of crude GAR-Per conjugate. Gar-Per genes, P. vulgaris, S. marcescens, P. fluoresconjugate, fractions 38 through 43, saved. cens, an Erwinia sp., and a Proteus sp. were
Within 5 weeks after the first injection, flagella-immunized rabbits responded with serum agglutination titers of 1:8,192 which were maintained for several weeks. During this period sera were collected. The IgG fractions were isolated by gel filtration, pooled, and lyophilized. When the partially purified IgG powder was dissolved in PBS (1 mg/ml), the solution had an agglutination titer of 1:32. GAR-Per. Conjugation of peroxidase (Per) to the IgG fraction of goat anti-rabbit IgG serum (GAR) was demonstrated by comparing the gelfiltration elution profile of crude GAR-Per conjugate (Fig. 4C) with the elution profiles of uncoupled GAR alone (Fig. 4A) and uncoupled GAR plus uncoupled Per (Fig. 4B). Molecular conjugates containing both antibody and enzyme would have a molecular weight of 190,000 or greater (150,000 for IgG plus 40,000 for peroxidase), a molecular size at or near the exclusion limit of Sephadex G-200. Therefore, samples containing antibody-peroxidase conjugates
IA 1-
0
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FIG. 5. Representative ELAT results with 25-mm cellulose-acetate membrane filters. Center membrane shows the template well pattern. Membranes I and II were developed in 3,3'-diaminobenzidine (-DAB-). Membranes III and IV were developed in benzidine (-B-). Membranes I and III were spotted as follows: spots 1-5 contained 107, 106, 105, 104, and 103 S. typhimurium cells, respectively; spot 6 contained 107 Proteus sp. cells; spot 7 contained 107 E. coli cells. Membranes II and IV were spotted as follows: spot 1 contained 107 S. typhimurium cells; spots 2-4 contained 107, 106, and 105 S. typhimurium cells, respectively, each mixed with 107 Proteus sp. cells; spots 5-7 contained 107, 106, and 105 S. typhimurium cells, respectively, each mixed with 107 E. coli cells.
tested. Specificity was lost when Difco poly-H antiserum was substituted for anti-S. typhimurium H antibody; C. freundii, E. coli, E. aerogenes, P. fluorescens, the Erwinia sp., and the Proteus sp. produced weak false-positive reactions. This may reflect the presence of nonspecific 0 antibody present in the Difco H-antiserum (7). Millipore (cellulose-nitrate) membranes were unsuitable for this methodology. Unreacted reagents could not be completely washed from membranes before exposure to substrate. This resulted in a background enzymatic product on membranes which masked the positive enzymatic product for S. typhimurium. When Gelman (cellulose-acetate) membranes were used, a blue reaction product with benzidine as substrate was preferred. Occasionally, insoluble material in enrichment broths, especially Mbroth inoculated with selenite-cystine broth, left a brown deposit on membranes which was confused with the brown enzymatic product which developed with 3,3'-diaminobenzidine. Nonspecific, false positives were not encoun-
tered when benzidine was the developing substrate. Tests in food products. Commercial pancake mix, NFDM, and commercial trout feed not inoculated with S. typhimurium were Salmonella negative by enrichment serology, cultural procedures, and the ELAT. At levels of 50, 10, and 2 S. typhimurium cells/g, each of the methods detected the presence of salmonellae in all food samples tested. In a comparison between enrichment serology and the ELAT on samples containing one S. typhimurium cell/10 g (95% confidence limits: 0.2 to 2.9), duplicate samples of NFDM were positive and pancake mix and fish feed were negative by both methods. For the ELAT, best results were obtained by spotting M-broth cultures showing the first sign of turbidity. In this study M-broth turbidity developed after approximately 4 h of incubation. This cell density was optimal for spotting membranes, and the spots contained sufficient numbers of S. typhimurium for detection. Because of the high cell density, 8-h M-broth cultures had to be diluted, usually 1:4, before spotting.
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DISCUSSION For a new Salmonella detection methodology to be acceptable, it must offer some advantage or improvement over existing methodologies. The ELAT appears to satisfy these criteria. The ELAT uses a filter membrane as a matrix for detection. A 47-mm membrane can be spotted with 14 or more broth enrichment samples, facilitating multisample analyses. The procedure requires little technical expertise or equipment and is relatively inexpensive to perform. In this study 100 mg of peroxidase coupled to antibody was sufficient to test over 10,000 Salmonella spots. Detection occurred through the amplification effect of the enzyme-substrate reaction, producing a macroscopically visible deposit of insoluble product on membranes spotted with broth cultures containing S. typhimurium. In contrast to the 5 x 107 salmonellae/ml needed for detection by enrichment serology or fluorescent-antibody techniques (18), the ELAT was sensitive to 105, and to a lesser extent 104, S. typhimurium cells/ml. When Sperber and Deibel's enrichment broths were used, the ELAT performed on M-broth cultures showing the first sign of turbidity (about 4 h) agreed with the results obtained by enrichment serology and cultural procedures on three food products containing low levels of S. typhimurium. The total analysis time was 48 h: 18 h of preenrichment, 24 h of selective enrichment, 4 h in Mbroth, a 2 h for the ELAT. The procedure demonstrated Salmonella specificity when pure H antisera free from 0 antibodies were used. False-positive results ocurred with Difco poly H antisera, probably as a result of the presence of nonspecific 0 antibodies. This may represent a limiting factor in the ELAT since a pool of pure H antisera for common Salmonella serotypes appears to be necessary before the method would be of value in the routine surveillance of Salmonella in foods. Both cellulose-acetate membranes and a benzidine substrate are recommended for the methodology. Cellulose-nitrate membranes produced unsatisfactory results because residual reactants which could not be completely removed resulted in background enzymatic product. False-positive results encountered with 3,3'-diaminobenzidine were eliminated by using benzidine as substrate. The resulting blue reaction product could only be interpreted as an enzymatic reaction product and could not be confused with nonenzymatic brown deposits from enrichment media.
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A chemical fixation step to prevent spotted cells from washing off membranes was not needed. Membranes which were air-dried after spotting produced results similar to membranes fixed in glutaraldehyde. This can be attributed to the mild washes employed in the methodology and the fixation inherent to air drying. The washes used were sufficient to remove unreacted reagents from membranes; thus, purification of crude GAR-Per was not necessary. This study demonstrated that enzyme-labeled antibodies can be used as a sensitive and specific methodology for macroscopic detection of Salmonella in mixed enrichment cultures. Further work using a polyvalent H antisera on many food samples is needed. A similar enzyme-labeled antibody method could be adapted for clinical analyses or in other systems where specific antigenic determinants are available for detection. ACKNOWLEDGMENTS This work was supported by the University of Idaho Agricultural Research Experiment Station under project 708-R670. LITERATURE CITED 1. Aleksic, S., and R. Rohde. 1972. The separation and
purification of Salmonella-Arizona H-antigens by DEAE-cellulose chromatography for the preparation and purification of diagnostic H-antisera with high titers and free of 0-antibodies. Ann. Inst. Pasteur Paris 123:363-370. 2. Association of Official Analytical Chemists. 1970. Official methods of analysis, 11th ed. Association of Official Analytical Chemists, Washington, D.C. 3. Avrameas, S. 1969. Coupling of enzymes to proteins with glutaraldehyde. Immunochemistry 6:43-52. 4. Barkata, J. A. 1968. Screening of feed components for Salmonella with polyvalent H agglutination. Appl. Microbiol. 16:1872-1874. 5. Edwards, D. R., and W. H. Ewing. 1972. Identification of Enterobacteriaceae, 3rd ed. Burgess Publishing Co., Minneapolis, Minn. 6. Elliot, R. P. (ed.). 1966. Bacteriological analytical manual. U.S. Food and Drug Administration, Washington, D.C. 7. Goepfert, J. M., and R. Hicks. 1969. Immunofluorescent staining of Salmonella species with flagella sera. Appl. Microbiol. 18:612-617. 8. Goldman, M. 1968. Fluorescent antibody methods, p. 94. Academic Press Inc., New York. 9. Gordon, M. A., and E. Lapa. 1971. Charcoal particle agglutination test for detection of antibody to Cryptococcus neoformas. Am. J. Clin. Pathol. 56:354-359. 10. Grahm, R. C., Jr., and M. J. Karnovsky. 1966. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural chemistry by a new technique. J. Histochem. Cytochem. 14:291-302. 11. Insalata, N. F., C. W. Mahnke, and W. G. Dunlap. 1974. Rapid, direct fluorescent antibody method for the detection of Salmonella in foods and feeds. Appl. Microbiol. 24:645-649. 12. Insalata, N. F., S. Schulte, and J. Berman. 1967. Immunofluorescent technique for detection of Salmo-
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nella in various foods. Appl. Microbiol. 15:1145-1149. 13. Mohr, H. K., H. C. Trenk, and M. Yeterian. 1970. Comparison of fluorescent antibodv methods and enrichment serology for the detection of Salmonella. Appl. Microbiol. 27:324-328. 14. Nakane, P. K. 1970. Classification of anterior pituitary cell types with immunoenzyme histochemistry. J. Histochem. Cytochem. 18:9-20. 15. Nakane, P. K., and G. B. Pierce, Jr. 1967. Enzymelabeled antibodies for the light and electron microscope localization of tissue antigens. J. Cell Biol. 33:307-317. 16. Reamer, R. H., and R. Hargrove. 1972. Twenty-fourhour immunofluorescence technique for the detection of Salmonella in nonfat dried milk. Appl. Microbiol. 23:78-81. 17. Rhodes, B. 1965. Liquid scintillation counting of radioiodine. Anal. Chem. 37:995. 18. Sperber, W. H., and R. H. Deibel. 1969. Accelerated procedure for Salmonella detection in dried foods and feeds involving only broth cultures and serological reactions. Appl. Microbiol. 17:553-539.
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Strafford, W. F., and D. A. Yphantis. 1972. Existence and inhibition of hydrolytic enzymes attacking paramyosin in myofibrillar extracts of Mercenaria mercenaria. Biochem. Biophys. Res. Commun. 49:848-845. 20. Straus, W. 1964. Factors affecting the cytochemical reaction of peroxidase with benzidine and the stability of the blue reaction product. J. Histochem. Cytochem. 12:462-468. 21. Ubertini, T., B. Wilkie, and F. Noronha. 1971. Use of 19.
horseradish peroxidase-labeled antibody for light and electron microscope localization of reovirus antigen. Appl. Microbiol. 21:534-538. 22. Weber, K., and M. Osborn. 1969. The reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406-4412. 23. Wicker, R., and S. Avrameas. 1969. Localization of virus antigens by enzyme-labeled antibodies. J. Gen. Virol. 4:465-468. 24. Worthington Biochemical Corp. 1972. Worthington enzyme manual. Worthington Biochemical Corp., Freehold, N.J.
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