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[41] Immunoassay of sulfate-reducing bacteria in environmental samples
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is intended as an introduction to these tools and as a source for initiating the discovery process with the literature cited. Acknowledgments This work was funded in part by grants from the National Institutes of Health (Grant No. GM41482) and the North Atlantic Treaty Organization (Grant No. 0404/88). Special thanks are due to Dr. Walter McRae, Assistant Vice President for Computing, for support funds and computer time on the high-performance machines.
[41] I m m u n o a s s a y o f S u l f a t e - R e d u c i n g B a c t e r i a in Environmental Samples By J.
MARTIN ODOM and RICHARD C. EBERSOLE
Introduction A method for immunoassay detection of sulfate-reducing bacteria has been developed specifically in response to the need for real-time estimations of sulfate-reducing bacteria in surface and subsurface oil and refining operations. This method involves modification and optimization of standard immunoassay principles for efficient capture and detection of an internal molecular marker for sulfate-reducing bacteria. Methods were also develoepd to obviate immunoassay interferences present in environmental samples. The procedures and devices described give order-ofmagnitude estimates of sulfate-reducing bacteria in environmental samples. Although this method was developed specifically for sulfate reducers, the strategy and methodologies can, in principle, be adapted for detection of other microorganisms from a variety of clinical or industrial environments. The need for a field test for sulfate-reducing bacteria can be found in the historical association of sulfate-reducing bacteria with oil souring, corrosion of underground metal and concrete structures, and plugging of oil-containing formations associated with oil exploration since its beginning.1 Although these consequences of microbial activity have been recognized for at least a century, the technology for detection and control of sulfate-reducing bacteria has not advanced since the early days of oil exploration. At that time, culture methods were developed that have i j. M. Odom, in "The Sulfate-ReducingBacteria: ContemporaryPerspectives" (J. M. Odom and R. S. Singleton,Jr., eds.), p. 189. Springer-Verlag,New York, 1993. METHODS IN ENZYMOLOGY, VOL. 243
Copyright © 1994by AcademicPress, Inc. All rights of reproduction in any form reserved.
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remained in use within the oil industry until the present time2 Culture methods for bacterial detection, particularly detection of sulfate reducers, suffer from the problems of incubation time (days to weeks) and uncertain specificity associated with a particular nutritional condition. Early detection and real-time monitoring of sulfate reducers for the purpose of assessing microbicide effectiveness, and sources and extent of bacterial contamination, are the principal driving forces behind development of this method. General Considerations for Immunoassay of Environmental Samples Choice of the analyte for detection is critical to test specificity. Immunoassays for particular groups of related bacteria require a conserved antigen unique to and present in all members of that group. For detection of a particular species, a species-specific antigen or epitope must be isolated. Taxonomic makers for immunoassay are ideally extracellular and thus freely accessible for antigen-antibody reaction. Antigenic properties of extracellular cytochromes c 3 and cell surface antigens from D e s u l f o v i b r i o spp. and D e s u l f o t o m a c u l u m spp. have been investigated by others with the conclusion that species or genus specificity prevented useful application of these components as markers for sulfate-reducing bacteria as a whole. 3-5 Species-specific antigens appear to be more common than broadly crossreactive antigens. This is probably due to the deep taxonomic divisions within this grouping with the result that the only trait many sulfate reducers have in common is the ability to reduce sulfate to hydrogen sulfide. 6 The enzymatic pathway of respiratory sulfate reduction consists of, at a minimum, the ATP-sulfurylase, adenosine-5'-phosphosulfate (APS) reductase, and bisulfite reductase. We chose APS reductase, an obligatory enzyme for sulfate reduction, as a likely taxonomic marker protein for all sulfate-reducing bacteria because of its restricted occurrence and high cellular concentration. 7 A complicating factor is the presence of the enzyme in some sulfide-oxidizing bacteria. Thus, one question to be addressed is whether sufficient antigenic dissimilarity exists beween APS reductases from sulfate reducers and sulfide oxidizers for the enzyme to be useful as a taxonomic marker for sulfate reducers. 2AmericanPetroleumInstitute (API), "RecommendedPractice for BiologicalAnalysisof Subsurface InjectionWaters," Second Ed. API, Dallas Division, 1982. 3A. Norqvist and R. Roffey,Appl. Environ. Microbiol. 50, 31 (1985). 4 R. Singleton,Jr., J. Denis, and L. L. Campbell,Arch. Microbiol. 139, 91 (1984). 5R. Singleton,Jr., J. Denis, and L. L. Campbell,Arch. Microbiol. 141, 195 (1985). 6 R. Devereauxand D. A. Stahl, in "The Sulfate-ReducingBacteria:ContemporaryPerspectives" (J. M. Odom and R. S. singleton,Jr., eds.), p. 131. Springer-Verlag, New York, 1993. 7 R. N. Bramlettand H. D. Peck, Jr., J. Biol. Chem. 250, 195 (1975).
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A number of operational constraints are also inherent in development of an immunoassay for field use. Definitive and relevant criteria for detection limit, incubation times, and specificity are crucial and should be targeted at the outset of the development process. Limits on test sensitivity and specificity are properties of the antibody reagents but test design and sample pretreatment may enhance these parameters significantly over that observed with reagents in a standard enzyme-linked immunosorbent assay (ELISA) microtiter plate format. Another constraint is the nature of the sample to be assayed. Detection of an antigen contained within a toxic or inhibitory environmental milieu poses a unique challenge in terms of sample clean-up and pretreatment. Field detection also imposes additional constraints on the immunoassay test format. A field test format is generally constrained to the use of nonradioactive procedures relying on visual or colorimetric readout. Furthermore, field detection requires simple sample processing, which enables rapid and efficient analyte capture. Field Immunoassay for Sulfate-Reducing Bacteria Field immunoassay components consist of the basic antibody reagents and the hardware for sample processing and performance of the analyses. Antibody reagents for field use can also be utilized in the laboratory microtiter plate format using standard ELISA procedures, s A formal description of the microtiter plate format is minimal and focus is on components, methods, and problems unique to the field test.
Overview: Immunoassay Components and Test Procedure Sample Pretreatment. Purification of bacteria from the environmental sample and removal of interfering substances are achieved by entrapment of bacteria in a diatomaceous earth (DE) matrix (Fig. 1). The diatomaceous earth and sample mixture are collected in a filter, washed to remove solutes, and then transferred to a reagent vial and resuspended in a buffer containing anti-APS reductase-alkaline phosphatase conjugate. Cell Lysis. Reaction between antigen (APS reductase) and anti-APS reductase-alkaline phosphatase conjugate occurs simultaneously on sonication of the diatomaceous earth-bacteria-conjugate mixture. Capture. Immobilization (Fig. 2) of the antigen-antibody conjugate complex is attained by passing the filtered, sonicated suspension through the capture bead. The capture bead consists of a glycidyl methacrylate8 p. Tijssen, in "Practice and Theory of Enzyme Immunoassays" (R. H. Burdon and P. H. van Knippenberg, eds.), p. 221. Elsevier, Amsterdam, and New York, 1985.
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SPECIAL TECHNIQUES
Sample ~Cap
Reagent Vial Re-suspension CellLysls
Sampl Vial e
A
~
Processed Sampl Fluid e
Waste
FIG. 1. Sample pretreatment. Diatomaceous earth (DE) is mixed with a volume of the sample to be assayed. The bacteria are entrapped in the DE matrix when the sample is expelled through a porous filter housed within the filter cap. The bacteria-DE matrix is then added to a reagent vial containing anti-APS reductase-alkaline phosphatase conjugate in test buffer. The cells are lysed by sonication and the soluble APS reductase-conjugate complex is expelled through a second filter cap for subsequent assay.
Sonlcetor
t/
PipetteDevice
Sample
~e EnzymeAntibody
istrat
Conjugate
Cell Lysis
Anatyte Captu[e
Detection
FIG. 2. Field immunoassay for sulfate-reducing bacteria. The environmental sample (pretreated or untreated) is lysed by sonication in the presence of anti-APS reductase-alkaline phosphatase conjugate. The APS reductase-conjugate complex is then captured by passage over a porous capture bead. The capture bead can be housed within a plastic pipette. After washing off excess conjugate,the amount of bound APS reductase-conjugate is visualized by addition ofa chromogenic substrate,which imparts a color to the capture bead proportional to the amount of APS reductase in the sample.
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grafted microporous plastic bead to which affinity-purified anti-APS reductase antibody has been covalently attached. Detection. Visualization of bound antigen is achieved by washing excess, unbound conjugate off of the capture bead with a wash buffer followed by exposure to a chromogenic alkaline phosphatase substrate (5-bromo-4-chloroindolyl phosphate). Color intensity, generated over a standardized time interval, will be proportional to the amount of bound APS reductase on the bead. Estimations of Cell Numbers. Quantitative estimations of sulfate-reducing bacteria present in the sample are derived by visual comparison of the color generated by the test sample with a standard curve relating color intensity to cell numbers (determined by microscopic counts) of a standard sulfate-reducing organism.
Preparation of Immunoassay Components
Growth of Bacteria Desulfovibrio desulfuricans G100A, a strain originally isolated from oil well production water, was used as a source of the APS reductase. 9 Adenosine-5'-phosphosulfate reductases from the following bacterial strains were required for specificity studies: Desulfomicrobium baculaturn, Desulfovibrio salexigens, Desulfosarcina variabilis, Desulfovibrio gigas, Desulfovibrio vulgaris, D. desulfuricans 27774, D. desuifuricans 13541, D. desulfuricans API, Desulfovibrio multispirans, Desulfotomaculure nigrificans, Desulfotomaculum orientis, Desulfotomaculum ruminis and Desulfobulbus propionicus, Thiobacillus denitrificans, Chromatium vinosum, Chlorobium thiosulfatophilum, Escherichia coli, and Streptomyces lividans. All organisms and cell extracts were grown and prepared as previously described. 10
Adenosine-5'-phosphosulfate Reductase Purification Antibody purification involves affinity chromatography using covalently bound APS reductase to extract purified antibody from crude antisera. It is critically important, particularly for a large antigen such as APS reductase (190 kDa), that its native configuration be conserved and 9 p. j. Weimer, M. J. van Kavelaar, C. B. Michel, and T. K. Ng, Appl. Environ. Microbiol. 54, 386 (1988). to j. M. Odom, K. Jessie, E. Knodel, and M. Emptage, Appl. Environ. Microbiol. 57, 727 (1991).
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denaturation be limited. Therefore a rapid, high-yield purification procedure is required to satisfy the aforementioned criteria. A cell-free crude extract is prepared from a suspension ofD. desulfuricans G100A (50 g of frozen cell paste) cells in 100 mM phosphate buffer (1 : 2.5, w/v). This suspension is lysed by passage through an SLM-Aminco French pressure cell (SLM-Aminco, Urbana, IL) at 20,000 lb/in2. Cell debris and unbroken cells are removed by centrifugation at 108,000 g for 1 hr. The supernatant from step 1 is adjusted to 30% of saturation with ammonium sulfate (grade III; Sigma Chemical Co., St. Louis, MO) and then centrifuged at 20,000 g for 20 min and the pellet discarded. The supernatant from this step is increased to 60% of saturation with ammonium sulfate. The pellet from the 60% saturation supernatant contains most of the APS reductase and is redissolved in 50 mM Tris [Tris(hydroxymethyl)aminomethane] (Sigma Chemical Co.) buffer, pH 7. This extract is then applied to a phenyl-Sepharose CL-4B (Sigma Chemical Co.) column (15 × 2.5 cm) equilibrated with 700 mM ammonium sulfate in 50 mM Tris, pH 7. The column is washed with 10 mM ammonium sulfate in 50 mM Tris, pH 7, and then the APS reductase elutes with a 50% (v/v) mixture of glycerol and 50 mM Tris, pH 7. DE-52 (Whatman Biosystems, Ltd., Maidstone, Kent, England) ion-exchange chromatography is used to remove the glycerol and concentrate the enzyme for gel filtration. The phenyl-Sepharose CL-4B eluate is applied directly to a Whatman DE-52 column (5 x 1.5 cm) followed by washes of 40 mM potassium phosphate buffer, pH 7, and then eluted with 100 mM potassium phosphate buffer, pH 7. Gel filtration on Sephacryl S-300 (2.5 × 30 cm) at 0.5 ml/ min in 50 mM Tris buffer, pH 7, results in an electrophoretically pure enzyme. The purification scheme is shown in Table I. The same purificaTABLE 1 PURIFICATION OF ADENOSINE-5'-PHOSPHOSULFATE REDUCTASE FROM Desulfovibrio desulfuricans G100A °
Step Crude extract Ammonium sulfate Phenyl-Sepharose DE-52 SephacryI-S300
Volume (ml)
Total units b
Units/ml
Protein (mg/ml)
Specific activity (units/mg)
120 63
1590 1123
13 18
37 25
0.36 0.71
50 12 30
712 438 360
14 36 12
7 18 3
2.0 2.0 4.0
a Courtesy American Society for Microbiology. b Micromoles of ferricyanide reduced per minute.
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tion scheme was used for APS reductases from other sulfate-reducing and sulfide-oxidizing bacteria. The key step in the purification is the high affinity of all APS reductases for phenyl-Sepharose CL-4B. The purified enzyme from D. desulfuricans G 100A is composed of Mr 70,000 and 20,000 subunits with a total molecular weight of 190,000. Subunit stoichiometry is unknown but the enzyme contains 0.74 mol of FAD per mole of enzyme, and 6.3 mol of nonheme iron and 6 mol of acid-labile sulfide per mole of enzyme. The enzyme was assayed spectrophotometrically by measuring AMP and sulfite-dependent ferricyanide reduction. 7
Antibody Production Antiserum to D. desulfuricans G100A APS reductase is generated by initial intradermal immunization of New Zealand White rabbits with 250 ~g each of enzyme in Freund's complete adjuvant. Subsequently the rabbits receive subcutaneous booster injections of 50/xg of enzyme every 30 days. At biweekly intervals, 20 ml of blood can be obtained from each rabbit for antisera production. Hazleton Research Products, Inc. (Denver, PA) performed all rabbit maintenance, immunizations, and bleeds. Antibody titer determinations are performed on antisera using standard microtiter plate ELISA methodology.8 Immulon II microtiter plates (Dynatech Laboratories, Alexandria, VA) are coated with APS reductase (1 mg/ ml) in buffer composed of 50 mM Tris, pH 7.5, and 100 mM sodium chloride overnight at 4°. Blocking of the plates against nonspecific adsorption is achieved by 2-hr incubation with the same buffer containing bovine serum albumin (1 mg/ml) and 0.1% (v/v) Tween 20 (Sigma Chemical Co.) (blocking buffer). Antisera or purified antibody dilutions are added to the plates in blocking buffer and incubated for 2 hr followed by a blocking buffer rinse. Goat anti-rabbit immunoglobulin G-alkaline phosphatase conjugate (Boehringer Mannheim, Indianapolis, IN) at 3000-fold dilution in blocking buffer is applied at 200/~l/well and incubated for 2 hr. Finally the plates are washed four times with blocking buffer and color development initiated with 200 ~l/well of p-nitrophenol phosphate [1 mg/ml Sigma 221 buffer (Sigma Chemical Co.)]. Color development proceeds for approximately 5 min, at which time color intensity is determined by absorbance at 410 nm. Microtiter plate ELISAs of antisera or antibody for specificity studies are carried out in a similar fashion as previously described.I°
Antibody Reagent Preparation The multisubunit nature of APS reductase presents special problems for using the enzyme in affinity purification of the antibody. Typically a
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covalently bound antigen is used to adsorb the specific antibody from crude antisera and then the antibody is eluted with chaotropic agents or low pH. Adenosine-5'-phosphosulfate reductase must first be dispersed into individual subunits before covalent binding to an affinity column matrix to preclude the possibility that subunit complexes may contribute a high percentage of noncovalently bound material to the column matrix. This noncovalent material will then be present as an antigen contaminant in the eluted antibody material. In our procedure the enzyme is pretreated by dispersing APS reductase (0.2 mg/ml) in a buffer consisting of 100 mM sodium bicarbonate and 100 mM n-octyl-/3-D-glucopyranoside (Sigma Chemical Co.), pH 8.3, for 1 hr at 37°. Cyanogen bromide-activated Sepharose 4B (Pharmacia, Piscataway, N J) is then added to the detergent-dispersed enzyme at a ratio of 2.7 mg of enzyme per gram of gel (dry weight) and allowed to incubate at 4 ° overnight with gentle mixing. The gel is then placed in a column and washed (overnight at 5 ml/min) with 10 mM sodium phosphate, 10 mM NaCI, and 20 mM n-octyl-/3-D-glucopyranoside (pH 7) buffer or until all traces of ELISA-detectable antigen are removed from the column effluent. This step is critically important for removal of noncovalently bound enzyme. For immunoadsorption of specific APS reductase antibody, titered antiserum is passed over the column at 5 ml/min. The column is then washed with 10 mM sodium phosphate, 10 mM sodium chloride (pH 7) buffer until all the residual nonadhering protein is removed as determined by a stable optical density at 280 nm. A wash buffer composed of 100 mM glycine (pH 4) and 100 mM NaCI is used to elute more tightly bound contaminating material from the column. The anti-APS reductase antibody is then eluted with 100 mM glycine, pH 2.5, and 100 mM NaC1; the pH of the eluate is immediately adjusted to pH 7 with an excess of I M potassium phosphate buffer, pH 7, on collection. Anti-APS reductase-alkaline phosphatase conjugate preparation is by a one-step glutaraldehyde conjugation procedure linking antibody to alkaline phosphatase. 11 The procedure utilizes alkaline phosphatase (Boehringer Mannheim) and antibody in a 2 : 1 (w/w) ratio. Purification of the conjugate away from unconjugated antibody or enzyme is performed by preparative-scale high-performance liquid chromatography using a BioSil SEC 250-5 (300 x 7.8 mm) column (Bio-Rad Laboratories, Richmond CA) preparative column in 100 mMpotassium phosphate buffer. Conjugate titer is determined by ELISA using Immulon II microtiter plates coated with APS reductase (1 mg/ml, 200/zl/well) and blocked in blocking buffer. ii p. Tijssen, in "Practice and Theory of Enzyme Immunoassays" (R. H. Burdon and P. H. van Knippenberg, eds.), p. 221. Elsevier, Amsterdam, New York, 1985.
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Dilutions of the conjugate are then plated directly on the antigen-coated plate and incubated for 2 hr. Color development is performed as described above for antibody titer. Conjugate synthesis is a process that is difficult to control precisely, thus the product may vary in its activity and final concentration. Optimization of the amount of this product to be used in the field assay is an empirical process that entails matching conjugate dilution in test buffer with the antibody capture bead such that a minimal blank and maximal sensitivity are obtained.
Antibody Solid-Phase Support: "Capture Bead" The surface properties of the porous plastic supports, used in the field test device for analyte capture, are important to facilitate antibody attachment and to minimize assay interferences that can result from oil fouling and nonspecific adsorption of the enzyme conjugate reagents to the surface of the solid support. We have employed electron beam graft polymerization to modify the surface properties of the plastic supports. 12 The grafting process enables different types of polymers to be chemically bonded to the surface of the plastic support. In this way a means is provided to alter the surface properties of the solid-phase support. Glycidyl methacrylate (GMA) is an attractive monomer for this application because the resulting graft GMA polymer provides reactive epoxide groups facilitating direct antibody coupling to the surface of the support via reactive amino groups on the antibody itself. Furthermore, the treatment both increases the quantity of antibody that can be attached to the support and suppresses binding of interfering substances. Grafting is accomplished by irradiating the porous plastic supports with high-energy electrons (3 MeV). This produces radicals throughout the plastic support. The radicals provide sites for initiating polymerization when the irradiated support is placed in contact with monomer such as glycidyl methacrylate.
Surface Actioation of Supports Bullet-shaped porous polyethylene beads (0.169-in. diameter, 0.162in. length) are purchased from Porex, Inc. (Fairburn, GA). The supports are constructed of interconnecting pores ranging in size from 20 to 40 /zm. Glycidyl methacrylate (>97% pure), the monomer used for surface modification, is obtained from Aldrich (Milwaukee, WI). Prior to use, a grafting monomer solution is prepared by dissolving 15% (v/v) GMA in reagent grade tertiary butanol at 40°. 12 H. R. Allcock and F. W. Lampe, in "Contemporary Polymer Chemistry," p. 203. PrenticeHall, Englewood Cliffs, New Jersey, 1981.
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Surface grafting is accomplished by placing the porous beads (100 gm) in a polyethylene bag containing 200 ml of the GMA grafting monomer solution. Air is then removed from the bag by introduction of argon gas. Excess argon is removed from the bag and the top closed by heat sealing. For irradiation, the bag is placed in a level tray and the beads spread out in a uniform layer over the interior of the bag. The bead-monomer mixture is then irradiated with a 4-Mrad dose of electrons (2.8 MeV) at a dose rate of 1.2 Mrads/sec. Following irradiation the bag is shaken and the polymerization reaction is allowed to proceed at room temperature for 4 hr at room temperature. To free the beads of homopolymer and residual monomer, methylene chloride (750 ml) is then introduced and allowed to equilibrate for 10 min. The beads are then washed three times with equal portions of methylene chloride. The grafted beads are then dried to constant weight under vacuum, using a slow stream of nitrogen. Analysis of the dried supports should indicate about 5 to 20% weight increase over untreated beads.
Antibody Attachment to Activated Capture Bead The treated bead supports (100) from the polymerization step are placed in a 25-rnl container fitted with a serum stopper, which is then evacuated to remove trapped air in the capture bead. An antibody solution (5 ml) containing affinity-purified anti-APS reductase (100/~g/ml) in 10 mM potassium phosphate buffer (pH 7)-10 mM sodium chloride (PBS buffer) is injected and the vial adjusted to atmospheric pressure. Evacuation and repressurization is repeated four times in order to promote contact of the interior surfaces of the bead with the antibody solution. This process facilitates contact between the antibody and the internal pores of the capture bead. The container is then rotated (10 revolutions/min) for 24 hr at 4°. Following equilibration, excess antibody is removed by aspiration. A blocking solution (10 ml) containing 0.1% bovine serum albumin (BSA) (Grade IV; Sigma Chemical Co.) in PBS buffer is introduced and the supports equilibrated with rotation at 4° in 15 min. The supports are washed four times with cold PBS buffer and stored in the cold in PBS containing 0.1% (w/v) sodium azide. The antibody-coated support beads can also be maintained in the dried state following freeze drying.
Pipette-Capture Bead Device The antibody-coated support beads are inserted into a pipette (3 ml, Cat. No. 233-9525; Bio-Rad, Inc.) by first removing the pipette tip and then inserting the bead to a point 0.5 cm from the neck of the pipette. By
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drawing the solution to be tested into the bulb of the pipette, and then expelling it, the pipette-bead assembly provides a convenient means of moving fluid through the bead. This format maximizes contact between the capture antibody on the surface of the porous bead and the solution to be tested.
Assay Buffers Samples to be assayed in the pipette-capture bead device should be in test buffer, which consists of Tris (50 mM, pH 7.5), sodium chloride (75 mM). SL-18 detergent (Olin Chemical Co., Stamford, CN), BSA (0.1%, w/v), and azide (0.02%, w/v). After the sample-conjugate mixture in test buffer has been cycled through the pipette-bead device, excess conjugate is washed out of the device with wash fluid [Tris (50 mM, pH 7.4) and azide (0.05%, w/v)]. The pH of the solution is adjusted to pH 7.4. For visualizing the amount of conjugate bound, a chromogenic substrate reagent consisting of 2.3 mM 5-bromo-4-chloroindolyl phosphate (Sigma Chemical Co.) in a 10% (v/v) 2-amino-2-methyl-l-propanol buffer at pH 10.2 containing 0.01% (w/v) MgCI2 (Sigma Chemical Co.) is drawn into the pipette-bead device.
Diatomaceous Earth Sample Processing Step This step relies on diatomaceous earth (DE) or a similar cell-entrapping matrix to facilitate collecting and washing the bacteria free of interfering solutes. In this process 10 ml of sample fluid is added to a sample collection vial containing a small amount of diatomaceous earth filtration medium (40-80 mg/ml sample). A filtration cap, containing a porous plastic frit (average pore size <10/zm) is then attached. The mixture is suspended by shaking the vial. The vial is then inverted and the suspended solids are allowed to settle into the filtration cap. The bottle is then squeezed to expel sample liquid to waste. During filtration, bacteria are entrapped in the filtration matrix and collected in the filtration cap. The bacteria are washed by reattaching the cap to a bottle containing 3.0 ml of wash fluid and squeezing the wash bottle. Cell lysis is achieved by 60-sec sonication cycles using a Branson model 450 sonifier equipped with a 1-mm probe tip. Celite (Celite Corp., Lompoc, CA) has been used successfully although effective cell recovery can be obtained with a number of different sorbent materials. Diatomaceous earths such as Kenite (200 or 700) (Witco Chemical Co., Chicago, IL), Celatom FW60 (Eagle Picher Co., Reno, NV), and Cuno M901 (AMF/Cuno Microfiltration Products, Meriden, CN) also give adequate recovery of sulfate-reducing bacteria.
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Test Performance
Specificity of Antibody Reagent Microtiter plate ELISA response of anti-D, desulfuricans G100A APS reductase crude antisera to crude extracts from sulfate-reducing bacteria, sulfide-oxidizing bacteria, as well as to control bacteria (which should not contain the enzyme) is shown in Table II. There appear to be three group-
T A B L E II CROSS-REACTIVITIES OF CRUDE EXTRACTS OF SULFATE-REDUCING BACTERIA AND SULFIDEOXIDIZING BACTERIA WITH ANTI-ADENOSINE-5 % PHOSPHOSULFATE REDUCTASE ANTIBODIES a
Organism
Strain G100A E L I S A b r e s p o n s e (%)
Desulfovibrio desulfuricans G100A A T C C 27774 API A T C C 13541
Desulfomicrobium baculatum Desulfovibrio vulgaris Desulfovibrio gigas Desulfovibrio salexigens Desulfovibrio multispirans
100 92 _+ 16 100 _+ 15 84 _+ 10 33 _+ 12 63 _+ 10 92 _+ I 1 76 _+ 9 69 _+ 15
Desulfotomaculum orientis Desulfotomaculum ruminis Desulfotomaculum nigrificans Desulfobulbus propion&us Desulfosarcina variabilis
48_+6 12_+3 22_+4 26_+6 21_+4
Thiobacillus denitrificans Chromatium oinosum Chlorobium thiosulfatophilum Escherichia coli Streptomyces lividans
8 +2 4 -+ 2 0 0 3 -+ 3
C o u r t e s y American Society for Microbiology. b The E L I S A r e s p o n s e at an optical density o f 410 n m as an average percentage of the Desulfovibrio desulfuricans G I 0 0 A r e s p o n s e over a range of from 6 to 600 ng o f crude extract protein per well for each organism.
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IMMUNOASSAY OF SULFATE-REDUCING BACTERIA
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ings of reactions, the most strongly cross-reactive (33-100% of G100A response) being mostly Desulfovibrio spp., including marine and freshwater strains. The second grouping consists of significantly less crossreactive (12-48% of G100A response) Desulfotomaculum, Desulfosarcina, and Desulfobulbus species. Cross-reactivity with Desulfotomaculum orientis is surprisingly high in light of the response with the other Desulfotomaculum species investigated. The third grouping consists of bacteria that lack APS reductase (E. coli and S. lividans), photosynthetic sulfide oxidizers (Chl. thiosulfatophilum and C. vinosum), and T. denitrificans. Of the non-sulfate-reducing bacteria investigated, the strongest response was consistently observed with T. denitrificans. It is anticipated from these results that antibody specificity will allow reasonable order-of-magnitude estimations of many Desulfovibrio spp., based on calibration of the response with cell suspensions of D. desulfuricans G100A, but will significantly underestimate the distantly related Desulfosarcina, Desulfobulbus, and Desulfotomaculum spp. A remedy for this would be the inclusion of antibodies to one or more of these organisms into the antibody capture and detection reagents. To see if these differences were actually due to the antigenic variation in the enzymes or to some other factor such as cellular enzyme content, enzyme extractability, or enzyme stability, enzymes from D. desulfuricans API. D. desulfuricans 27774, and Desulfomicrobium baculatum, D. vulgaris, Desulfotomaculum orientis, and T. denitrificans were purified to comparable specific activity and assayed by standard ELISA microtiter plate methodology (Table III). The data show that the purified enzyme reactivities paralleled crude cell lysate reactivities with the exception of Desulfotomaculum orientis, which gave essentially 100% of D. desulfuricans G100A response, suggesting that variations in response are due to antigenic differences in the enzymes themselves.
Sample Pretreatment Environmental samples may contain high concentrations of solid sediments, colloids, emulsions, soluble and insoluble salts, and industrial chemicals that can prevent or invalidate the immunoassay. To remove these potential interferences, sample processing is required prior to immunoassay. This is useful both to concentrate the small numbers of organisms found in field sample materials and to remove interfering materials that prevent analysis or cause inaccurate test results. Generally, sample treatment processes can involve centrifugation, membrane filtration or chemical precipitation; however, these procedures require laboratory facilities
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SPECIAL TECHNIQUES TABLE III SPECIFIC ACTIVITIES AND CROsS-REACTIVITIES OF ENZYMES TO ANTI-ADENOSINE-St-PHosPHOSULFATEREDUCTASE POLYCLONAL ANTIBODIESa Strain G100A ELISA response (%)b
activityC
Desulfomicrobium baculatum
100 84 57 38
3.5 2.0 2.0 0.8
Desulfovibrio vulgaris Desulfotomaculum orientis Thiobacillus denitrificans
76 100 8
1.8 3.0 2.3
Organism
Specific
Desulfovibrio desulfuricans G100A API ATCC 27774
Courtesy American Society for Microbiology. b Purified protein, 240 ng per well. c Micromoles of ferricyanide reduced per minute per milligram of protein.
and are not readily accomplished in the field. We have found that bacteria can be efficiently entrapped in and recovered from small filters containing a diatomaceous earth (DE) as the entrapping matrix. Microorganisms can be effectively recovered over a wide range of cell concentrations typically encountered in environmental samples as shown in Table IV. In this example, production water from an oil well (courtesy of Conoco, Inc.) is seeded with varying concentrations ofD. desulfuricans TABLE IV SAMPLE PRETREATMENT CELL RECOVERY EFFICIENCY VERSUS SAMPLE CELL DENSITY OF Desulfooibrio desalfuricans GI00A Response (OD410) Cells/ml (added) 0.5 0.5 0.5 0.5 0
× x x x
107 106 105 104
Tris buffer ~
DE b
Filtration c
1.70 0.80 0.16 0.05 0.04
1.80 0.80 0.20 0.08 0.07
1.60 0.70 0.12 0.04 0.08
Control, no treatment. b Diatomaceous earth (Celite) at 80 mg/ml sample fluid. c Membrane filtration.
a
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using a Petroff-Hauser cell counter such that dilutions up to 108 cells/ml are available for testing. Figure 3 shows the field immunoassay response to standard cell dilutions of D. desulfuricans G100A. The following steps and incubation times were employed. 1. Cell suspensions (10 ml) are collected by diatomaceous earth pretreatment and washed free of residual medium with wash fluid. 2. The diatomaceous earth-bacteria mixture is then mixed with 1.5 ml of test buffer containing conjugate and sonicated for 30 sec. The soluble APS reductase-conjugate complex generated in this step is removed from the diatomaceous earth matrix by filtration. 3. The filtrate from step 2 is passed through the antibody-coated microporous bead-pipette device four times. The bead-pipette device is rinsed four times with wash fluid. 4. For the purpose of colorimetric quantitation, the beads are then removed from the pipette and placed in individual microtiter plate wells containing p-nitrophenol phosphate at 1 mg/ml in Sigma 221 buffer and allowed to incubate for 15 min before quantitation of the color formed. In an actual field assay a solution of 5-bromo-4-chloroindolyl phosphate is drawn directly into the bead-pipette device and cell density is estimated visually by comparison of bead color intensity with a calibrated color
2.0-
1.5. E eo
1.o0 0.5.
o . o
.
0
1
.
.
.
.
.
.
.
.
.
.
2 3 4 5 LOG Cell Density
.
6
7
FIG. 3. Response of field immunoassay assay to different concentrations of D. desulfuricans G100A. Response was numerically quantitated by removal of the capture bead from the pipette for color development in a microtiter plate well. (Courtesy National Association of Corrosion Engineers.)
[41]
IMMUNOASSAY OF SULFATE-REDUCING BACTERIA
623
chart relating color intensity to cell density of D. desulfuricans G100A. The curve in Fig. 3 relates cell numbers to optical density at 410 nm; note that the limit of detection is close to 10 4 cells/ml. To determine the extent of underestimation of poorly cross-reactive strains as well as cross-reactivity with a strong responding strain, D. desulfuricans API (100% o f D . desulfuricans G100A response) and Desulfomicrobium baculatum ( - 3 0 - 4 0 % of D. desulfuricans G100A response) are tested to check the correlation between actual cell counts and those determined by comparison with the colorimetric response by D. desulfuricans G100A. Field immunoassays were performed as described above for the D. desulfuricans G100A strain. Table V shows almost a 100-fold underestimation for cell numbers for the Desulfomicrobium baculatum strain even though the crude extract and purified enzyme ELISA data indicated only a 2- to 3-fold difference in response under equilibrium conditions. There is generally much less than an order of magnitude difference in cell numbers for the D. desulfuricans API strain. More importantly, the detection limit appears to be between 10 3 and 104 for D. desulfuricans API and 104 and 105 for Desulfomicrobium baculatum. It is important to emphasize that the field immunoassay is a short time course nonequilibrium assay as opposed to the microtiter plate assay, which is essentially TABLE V RESPONSE OF FIELD TEST FORMAT TO CELLS OF Desulfovibrio desulfuricans API AND Desulfomicrobium baculatum AS DETERMINED BY COLORIMETRIC RESPONSE RELATIVE TO
Desulfovibrio desulfuricans GI00A Cell density by immunoassay b Actual cell density a
D. desulfuricans API
0
10z 103 104 105 106 107 108
2 2 9 2 5
0 0 0 × 103 x 105 x 106 × 107 X 107
Dsm. baculatum
6 6 4 9 4
0 0 0 × 102 x 102 × 104 × 104 × 106
Cell density as determined by microscopic counts using a PetroffHauser counter. b Cell density as determined by rapid immunoassay and comparison of color response with D.desulfuricans G100A standard response curve.
624
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an equilibrium assay. The nonequilibrium format apepars to be more sensitive to differences in affinity between antibody and the different APS reductases. However, we have found, using the identical reagents in the microtiter plate format, that the detection limit for D. desulfuricans G 100A is 105 to 106 cells/ml after a total incubation time of 2 hr as opposed to 2-3 min for the capture-bead format. Concluding Remarks We describe a method for estimating cell numbers of sulfate-reducing bacteria in crude environmental samples. There are two unique and key elements to this method: (1) the sample pretreatment step, which isolates the bacteria and removes soluble interferences, and (2) the antibodycoated microporous capture-bead device, which enables rapid and efficient capture of the antigen from the sample. At present antibody specificity restricts the sensitivity of the test to Desulfovibrio spp., however, inclusion of antibodies to APS reductases from other genera into the antibody reagent should expand the overall specificity of the test.
IMMUNOASSAY OF SULFATE-REDUCING BACTERIA
607
is intended as an introduction to these tools and as a source for initiating the discovery process with the literature cited. Acknowledgments This work was funded in part by grants from the National Institutes of Health (Grant No. GM41482) and the North Atlantic Treaty Organization (Grant No. 0404/88). Special thanks are due to Dr. Walter McRae, Assistant Vice President for Computing, for support funds and computer time on the high-performance machines.
[41] I m m u n o a s s a y o f S u l f a t e - R e d u c i n g B a c t e r i a in Environmental Samples By J.
MARTIN ODOM and RICHARD C. EBERSOLE
Introduction A method for immunoassay detection of sulfate-reducing bacteria has been developed specifically in response to the need for real-time estimations of sulfate-reducing bacteria in surface and subsurface oil and refining operations. This method involves modification and optimization of standard immunoassay principles for efficient capture and detection of an internal molecular marker for sulfate-reducing bacteria. Methods were also develoepd to obviate immunoassay interferences present in environmental samples. The procedures and devices described give order-ofmagnitude estimates of sulfate-reducing bacteria in environmental samples. Although this method was developed specifically for sulfate reducers, the strategy and methodologies can, in principle, be adapted for detection of other microorganisms from a variety of clinical or industrial environments. The need for a field test for sulfate-reducing bacteria can be found in the historical association of sulfate-reducing bacteria with oil souring, corrosion of underground metal and concrete structures, and plugging of oil-containing formations associated with oil exploration since its beginning.1 Although these consequences of microbial activity have been recognized for at least a century, the technology for detection and control of sulfate-reducing bacteria has not advanced since the early days of oil exploration. At that time, culture methods were developed that have i j. M. Odom, in "The Sulfate-ReducingBacteria: ContemporaryPerspectives" (J. M. Odom and R. S. Singleton,Jr., eds.), p. 189. Springer-Verlag,New York, 1993. METHODS IN ENZYMOLOGY, VOL. 243
Copyright © 1994by AcademicPress, Inc. All rights of reproduction in any form reserved.
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remained in use within the oil industry until the present time2 Culture methods for bacterial detection, particularly detection of sulfate reducers, suffer from the problems of incubation time (days to weeks) and uncertain specificity associated with a particular nutritional condition. Early detection and real-time monitoring of sulfate reducers for the purpose of assessing microbicide effectiveness, and sources and extent of bacterial contamination, are the principal driving forces behind development of this method. General Considerations for Immunoassay of Environmental Samples Choice of the analyte for detection is critical to test specificity. Immunoassays for particular groups of related bacteria require a conserved antigen unique to and present in all members of that group. For detection of a particular species, a species-specific antigen or epitope must be isolated. Taxonomic makers for immunoassay are ideally extracellular and thus freely accessible for antigen-antibody reaction. Antigenic properties of extracellular cytochromes c 3 and cell surface antigens from D e s u l f o v i b r i o spp. and D e s u l f o t o m a c u l u m spp. have been investigated by others with the conclusion that species or genus specificity prevented useful application of these components as markers for sulfate-reducing bacteria as a whole. 3-5 Species-specific antigens appear to be more common than broadly crossreactive antigens. This is probably due to the deep taxonomic divisions within this grouping with the result that the only trait many sulfate reducers have in common is the ability to reduce sulfate to hydrogen sulfide. 6 The enzymatic pathway of respiratory sulfate reduction consists of, at a minimum, the ATP-sulfurylase, adenosine-5'-phosphosulfate (APS) reductase, and bisulfite reductase. We chose APS reductase, an obligatory enzyme for sulfate reduction, as a likely taxonomic marker protein for all sulfate-reducing bacteria because of its restricted occurrence and high cellular concentration. 7 A complicating factor is the presence of the enzyme in some sulfide-oxidizing bacteria. Thus, one question to be addressed is whether sufficient antigenic dissimilarity exists beween APS reductases from sulfate reducers and sulfide oxidizers for the enzyme to be useful as a taxonomic marker for sulfate reducers. 2AmericanPetroleumInstitute (API), "RecommendedPractice for BiologicalAnalysisof Subsurface InjectionWaters," Second Ed. API, Dallas Division, 1982. 3A. Norqvist and R. Roffey,Appl. Environ. Microbiol. 50, 31 (1985). 4 R. Singleton,Jr., J. Denis, and L. L. Campbell,Arch. Microbiol. 139, 91 (1984). 5R. Singleton,Jr., J. Denis, and L. L. Campbell,Arch. Microbiol. 141, 195 (1985). 6 R. Devereauxand D. A. Stahl, in "The Sulfate-ReducingBacteria:ContemporaryPerspectives" (J. M. Odom and R. S. singleton,Jr., eds.), p. 131. Springer-Verlag, New York, 1993. 7 R. N. Bramlettand H. D. Peck, Jr., J. Biol. Chem. 250, 195 (1975).
[41]
IMMUNOASSAY OF SULFATE-REDUCING BACTERIA
609
A number of operational constraints are also inherent in development of an immunoassay for field use. Definitive and relevant criteria for detection limit, incubation times, and specificity are crucial and should be targeted at the outset of the development process. Limits on test sensitivity and specificity are properties of the antibody reagents but test design and sample pretreatment may enhance these parameters significantly over that observed with reagents in a standard enzyme-linked immunosorbent assay (ELISA) microtiter plate format. Another constraint is the nature of the sample to be assayed. Detection of an antigen contained within a toxic or inhibitory environmental milieu poses a unique challenge in terms of sample clean-up and pretreatment. Field detection also imposes additional constraints on the immunoassay test format. A field test format is generally constrained to the use of nonradioactive procedures relying on visual or colorimetric readout. Furthermore, field detection requires simple sample processing, which enables rapid and efficient analyte capture. Field Immunoassay for Sulfate-Reducing Bacteria Field immunoassay components consist of the basic antibody reagents and the hardware for sample processing and performance of the analyses. Antibody reagents for field use can also be utilized in the laboratory microtiter plate format using standard ELISA procedures, s A formal description of the microtiter plate format is minimal and focus is on components, methods, and problems unique to the field test.
Overview: Immunoassay Components and Test Procedure Sample Pretreatment. Purification of bacteria from the environmental sample and removal of interfering substances are achieved by entrapment of bacteria in a diatomaceous earth (DE) matrix (Fig. 1). The diatomaceous earth and sample mixture are collected in a filter, washed to remove solutes, and then transferred to a reagent vial and resuspended in a buffer containing anti-APS reductase-alkaline phosphatase conjugate. Cell Lysis. Reaction between antigen (APS reductase) and anti-APS reductase-alkaline phosphatase conjugate occurs simultaneously on sonication of the diatomaceous earth-bacteria-conjugate mixture. Capture. Immobilization (Fig. 2) of the antigen-antibody conjugate complex is attained by passing the filtered, sonicated suspension through the capture bead. The capture bead consists of a glycidyl methacrylate8 p. Tijssen, in "Practice and Theory of Enzyme Immunoassays" (R. H. Burdon and P. H. van Knippenberg, eds.), p. 221. Elsevier, Amsterdam, and New York, 1985.
610
[41]
SPECIAL TECHNIQUES
Sample ~Cap
Reagent Vial Re-suspension CellLysls
Sampl Vial e
A
~
Processed Sampl Fluid e
Waste
FIG. 1. Sample pretreatment. Diatomaceous earth (DE) is mixed with a volume of the sample to be assayed. The bacteria are entrapped in the DE matrix when the sample is expelled through a porous filter housed within the filter cap. The bacteria-DE matrix is then added to a reagent vial containing anti-APS reductase-alkaline phosphatase conjugate in test buffer. The cells are lysed by sonication and the soluble APS reductase-conjugate complex is expelled through a second filter cap for subsequent assay.
Sonlcetor
t/
PipetteDevice
Sample
~e EnzymeAntibody
istrat
Conjugate
Cell Lysis
Anatyte Captu[e
Detection
FIG. 2. Field immunoassay for sulfate-reducing bacteria. The environmental sample (pretreated or untreated) is lysed by sonication in the presence of anti-APS reductase-alkaline phosphatase conjugate. The APS reductase-conjugate complex is then captured by passage over a porous capture bead. The capture bead can be housed within a plastic pipette. After washing off excess conjugate,the amount of bound APS reductase-conjugate is visualized by addition ofa chromogenic substrate,which imparts a color to the capture bead proportional to the amount of APS reductase in the sample.
[41]
IMMUNOASSAY OF SULFATE-REDUCING BACTERIA
611
grafted microporous plastic bead to which affinity-purified anti-APS reductase antibody has been covalently attached. Detection. Visualization of bound antigen is achieved by washing excess, unbound conjugate off of the capture bead with a wash buffer followed by exposure to a chromogenic alkaline phosphatase substrate (5-bromo-4-chloroindolyl phosphate). Color intensity, generated over a standardized time interval, will be proportional to the amount of bound APS reductase on the bead. Estimations of Cell Numbers. Quantitative estimations of sulfate-reducing bacteria present in the sample are derived by visual comparison of the color generated by the test sample with a standard curve relating color intensity to cell numbers (determined by microscopic counts) of a standard sulfate-reducing organism.
Preparation of Immunoassay Components
Growth of Bacteria Desulfovibrio desulfuricans G100A, a strain originally isolated from oil well production water, was used as a source of the APS reductase. 9 Adenosine-5'-phosphosulfate reductases from the following bacterial strains were required for specificity studies: Desulfomicrobium baculaturn, Desulfovibrio salexigens, Desulfosarcina variabilis, Desulfovibrio gigas, Desulfovibrio vulgaris, D. desulfuricans 27774, D. desuifuricans 13541, D. desulfuricans API, Desulfovibrio multispirans, Desulfotomaculure nigrificans, Desulfotomaculum orientis, Desulfotomaculum ruminis and Desulfobulbus propionicus, Thiobacillus denitrificans, Chromatium vinosum, Chlorobium thiosulfatophilum, Escherichia coli, and Streptomyces lividans. All organisms and cell extracts were grown and prepared as previously described. 10
Adenosine-5'-phosphosulfate Reductase Purification Antibody purification involves affinity chromatography using covalently bound APS reductase to extract purified antibody from crude antisera. It is critically important, particularly for a large antigen such as APS reductase (190 kDa), that its native configuration be conserved and 9 p. j. Weimer, M. J. van Kavelaar, C. B. Michel, and T. K. Ng, Appl. Environ. Microbiol. 54, 386 (1988). to j. M. Odom, K. Jessie, E. Knodel, and M. Emptage, Appl. Environ. Microbiol. 57, 727 (1991).
612
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[41]
denaturation be limited. Therefore a rapid, high-yield purification procedure is required to satisfy the aforementioned criteria. A cell-free crude extract is prepared from a suspension ofD. desulfuricans G100A (50 g of frozen cell paste) cells in 100 mM phosphate buffer (1 : 2.5, w/v). This suspension is lysed by passage through an SLM-Aminco French pressure cell (SLM-Aminco, Urbana, IL) at 20,000 lb/in2. Cell debris and unbroken cells are removed by centrifugation at 108,000 g for 1 hr. The supernatant from step 1 is adjusted to 30% of saturation with ammonium sulfate (grade III; Sigma Chemical Co., St. Louis, MO) and then centrifuged at 20,000 g for 20 min and the pellet discarded. The supernatant from this step is increased to 60% of saturation with ammonium sulfate. The pellet from the 60% saturation supernatant contains most of the APS reductase and is redissolved in 50 mM Tris [Tris(hydroxymethyl)aminomethane] (Sigma Chemical Co.) buffer, pH 7. This extract is then applied to a phenyl-Sepharose CL-4B (Sigma Chemical Co.) column (15 × 2.5 cm) equilibrated with 700 mM ammonium sulfate in 50 mM Tris, pH 7. The column is washed with 10 mM ammonium sulfate in 50 mM Tris, pH 7, and then the APS reductase elutes with a 50% (v/v) mixture of glycerol and 50 mM Tris, pH 7. DE-52 (Whatman Biosystems, Ltd., Maidstone, Kent, England) ion-exchange chromatography is used to remove the glycerol and concentrate the enzyme for gel filtration. The phenyl-Sepharose CL-4B eluate is applied directly to a Whatman DE-52 column (5 x 1.5 cm) followed by washes of 40 mM potassium phosphate buffer, pH 7, and then eluted with 100 mM potassium phosphate buffer, pH 7. Gel filtration on Sephacryl S-300 (2.5 × 30 cm) at 0.5 ml/ min in 50 mM Tris buffer, pH 7, results in an electrophoretically pure enzyme. The purification scheme is shown in Table I. The same purificaTABLE 1 PURIFICATION OF ADENOSINE-5'-PHOSPHOSULFATE REDUCTASE FROM Desulfovibrio desulfuricans G100A °
Step Crude extract Ammonium sulfate Phenyl-Sepharose DE-52 SephacryI-S300
Volume (ml)
Total units b
Units/ml
Protein (mg/ml)
Specific activity (units/mg)
120 63
1590 1123
13 18
37 25
0.36 0.71
50 12 30
712 438 360
14 36 12
7 18 3
2.0 2.0 4.0
a Courtesy American Society for Microbiology. b Micromoles of ferricyanide reduced per minute.
[41]
IMMUNOASSAY OF SULFATE-REDUCING BACTERIA
613
tion scheme was used for APS reductases from other sulfate-reducing and sulfide-oxidizing bacteria. The key step in the purification is the high affinity of all APS reductases for phenyl-Sepharose CL-4B. The purified enzyme from D. desulfuricans G 100A is composed of Mr 70,000 and 20,000 subunits with a total molecular weight of 190,000. Subunit stoichiometry is unknown but the enzyme contains 0.74 mol of FAD per mole of enzyme, and 6.3 mol of nonheme iron and 6 mol of acid-labile sulfide per mole of enzyme. The enzyme was assayed spectrophotometrically by measuring AMP and sulfite-dependent ferricyanide reduction. 7
Antibody Production Antiserum to D. desulfuricans G100A APS reductase is generated by initial intradermal immunization of New Zealand White rabbits with 250 ~g each of enzyme in Freund's complete adjuvant. Subsequently the rabbits receive subcutaneous booster injections of 50/xg of enzyme every 30 days. At biweekly intervals, 20 ml of blood can be obtained from each rabbit for antisera production. Hazleton Research Products, Inc. (Denver, PA) performed all rabbit maintenance, immunizations, and bleeds. Antibody titer determinations are performed on antisera using standard microtiter plate ELISA methodology.8 Immulon II microtiter plates (Dynatech Laboratories, Alexandria, VA) are coated with APS reductase (1 mg/ ml) in buffer composed of 50 mM Tris, pH 7.5, and 100 mM sodium chloride overnight at 4°. Blocking of the plates against nonspecific adsorption is achieved by 2-hr incubation with the same buffer containing bovine serum albumin (1 mg/ml) and 0.1% (v/v) Tween 20 (Sigma Chemical Co.) (blocking buffer). Antisera or purified antibody dilutions are added to the plates in blocking buffer and incubated for 2 hr followed by a blocking buffer rinse. Goat anti-rabbit immunoglobulin G-alkaline phosphatase conjugate (Boehringer Mannheim, Indianapolis, IN) at 3000-fold dilution in blocking buffer is applied at 200/~l/well and incubated for 2 hr. Finally the plates are washed four times with blocking buffer and color development initiated with 200 ~l/well of p-nitrophenol phosphate [1 mg/ml Sigma 221 buffer (Sigma Chemical Co.)]. Color development proceeds for approximately 5 min, at which time color intensity is determined by absorbance at 410 nm. Microtiter plate ELISAs of antisera or antibody for specificity studies are carried out in a similar fashion as previously described.I°
Antibody Reagent Preparation The multisubunit nature of APS reductase presents special problems for using the enzyme in affinity purification of the antibody. Typically a
614
SPECIALTECHNIQUES
[41]
covalently bound antigen is used to adsorb the specific antibody from crude antisera and then the antibody is eluted with chaotropic agents or low pH. Adenosine-5'-phosphosulfate reductase must first be dispersed into individual subunits before covalent binding to an affinity column matrix to preclude the possibility that subunit complexes may contribute a high percentage of noncovalently bound material to the column matrix. This noncovalent material will then be present as an antigen contaminant in the eluted antibody material. In our procedure the enzyme is pretreated by dispersing APS reductase (0.2 mg/ml) in a buffer consisting of 100 mM sodium bicarbonate and 100 mM n-octyl-/3-D-glucopyranoside (Sigma Chemical Co.), pH 8.3, for 1 hr at 37°. Cyanogen bromide-activated Sepharose 4B (Pharmacia, Piscataway, N J) is then added to the detergent-dispersed enzyme at a ratio of 2.7 mg of enzyme per gram of gel (dry weight) and allowed to incubate at 4 ° overnight with gentle mixing. The gel is then placed in a column and washed (overnight at 5 ml/min) with 10 mM sodium phosphate, 10 mM NaCI, and 20 mM n-octyl-/3-D-glucopyranoside (pH 7) buffer or until all traces of ELISA-detectable antigen are removed from the column effluent. This step is critically important for removal of noncovalently bound enzyme. For immunoadsorption of specific APS reductase antibody, titered antiserum is passed over the column at 5 ml/min. The column is then washed with 10 mM sodium phosphate, 10 mM sodium chloride (pH 7) buffer until all the residual nonadhering protein is removed as determined by a stable optical density at 280 nm. A wash buffer composed of 100 mM glycine (pH 4) and 100 mM NaCI is used to elute more tightly bound contaminating material from the column. The anti-APS reductase antibody is then eluted with 100 mM glycine, pH 2.5, and 100 mM NaC1; the pH of the eluate is immediately adjusted to pH 7 with an excess of I M potassium phosphate buffer, pH 7, on collection. Anti-APS reductase-alkaline phosphatase conjugate preparation is by a one-step glutaraldehyde conjugation procedure linking antibody to alkaline phosphatase. 11 The procedure utilizes alkaline phosphatase (Boehringer Mannheim) and antibody in a 2 : 1 (w/w) ratio. Purification of the conjugate away from unconjugated antibody or enzyme is performed by preparative-scale high-performance liquid chromatography using a BioSil SEC 250-5 (300 x 7.8 mm) column (Bio-Rad Laboratories, Richmond CA) preparative column in 100 mMpotassium phosphate buffer. Conjugate titer is determined by ELISA using Immulon II microtiter plates coated with APS reductase (1 mg/ml, 200/zl/well) and blocked in blocking buffer. ii p. Tijssen, in "Practice and Theory of Enzyme Immunoassays" (R. H. Burdon and P. H. van Knippenberg, eds.), p. 221. Elsevier, Amsterdam, New York, 1985.
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IMMUNOASSAY OF SULFATE-REDUCING BACTERIA
615
Dilutions of the conjugate are then plated directly on the antigen-coated plate and incubated for 2 hr. Color development is performed as described above for antibody titer. Conjugate synthesis is a process that is difficult to control precisely, thus the product may vary in its activity and final concentration. Optimization of the amount of this product to be used in the field assay is an empirical process that entails matching conjugate dilution in test buffer with the antibody capture bead such that a minimal blank and maximal sensitivity are obtained.
Antibody Solid-Phase Support: "Capture Bead" The surface properties of the porous plastic supports, used in the field test device for analyte capture, are important to facilitate antibody attachment and to minimize assay interferences that can result from oil fouling and nonspecific adsorption of the enzyme conjugate reagents to the surface of the solid support. We have employed electron beam graft polymerization to modify the surface properties of the plastic supports. 12 The grafting process enables different types of polymers to be chemically bonded to the surface of the plastic support. In this way a means is provided to alter the surface properties of the solid-phase support. Glycidyl methacrylate (GMA) is an attractive monomer for this application because the resulting graft GMA polymer provides reactive epoxide groups facilitating direct antibody coupling to the surface of the support via reactive amino groups on the antibody itself. Furthermore, the treatment both increases the quantity of antibody that can be attached to the support and suppresses binding of interfering substances. Grafting is accomplished by irradiating the porous plastic supports with high-energy electrons (3 MeV). This produces radicals throughout the plastic support. The radicals provide sites for initiating polymerization when the irradiated support is placed in contact with monomer such as glycidyl methacrylate.
Surface Actioation of Supports Bullet-shaped porous polyethylene beads (0.169-in. diameter, 0.162in. length) are purchased from Porex, Inc. (Fairburn, GA). The supports are constructed of interconnecting pores ranging in size from 20 to 40 /zm. Glycidyl methacrylate (>97% pure), the monomer used for surface modification, is obtained from Aldrich (Milwaukee, WI). Prior to use, a grafting monomer solution is prepared by dissolving 15% (v/v) GMA in reagent grade tertiary butanol at 40°. 12 H. R. Allcock and F. W. Lampe, in "Contemporary Polymer Chemistry," p. 203. PrenticeHall, Englewood Cliffs, New Jersey, 1981.
616
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Surface grafting is accomplished by placing the porous beads (100 gm) in a polyethylene bag containing 200 ml of the GMA grafting monomer solution. Air is then removed from the bag by introduction of argon gas. Excess argon is removed from the bag and the top closed by heat sealing. For irradiation, the bag is placed in a level tray and the beads spread out in a uniform layer over the interior of the bag. The bead-monomer mixture is then irradiated with a 4-Mrad dose of electrons (2.8 MeV) at a dose rate of 1.2 Mrads/sec. Following irradiation the bag is shaken and the polymerization reaction is allowed to proceed at room temperature for 4 hr at room temperature. To free the beads of homopolymer and residual monomer, methylene chloride (750 ml) is then introduced and allowed to equilibrate for 10 min. The beads are then washed three times with equal portions of methylene chloride. The grafted beads are then dried to constant weight under vacuum, using a slow stream of nitrogen. Analysis of the dried supports should indicate about 5 to 20% weight increase over untreated beads.
Antibody Attachment to Activated Capture Bead The treated bead supports (100) from the polymerization step are placed in a 25-rnl container fitted with a serum stopper, which is then evacuated to remove trapped air in the capture bead. An antibody solution (5 ml) containing affinity-purified anti-APS reductase (100/~g/ml) in 10 mM potassium phosphate buffer (pH 7)-10 mM sodium chloride (PBS buffer) is injected and the vial adjusted to atmospheric pressure. Evacuation and repressurization is repeated four times in order to promote contact of the interior surfaces of the bead with the antibody solution. This process facilitates contact between the antibody and the internal pores of the capture bead. The container is then rotated (10 revolutions/min) for 24 hr at 4°. Following equilibration, excess antibody is removed by aspiration. A blocking solution (10 ml) containing 0.1% bovine serum albumin (BSA) (Grade IV; Sigma Chemical Co.) in PBS buffer is introduced and the supports equilibrated with rotation at 4° in 15 min. The supports are washed four times with cold PBS buffer and stored in the cold in PBS containing 0.1% (w/v) sodium azide. The antibody-coated support beads can also be maintained in the dried state following freeze drying.
Pipette-Capture Bead Device The antibody-coated support beads are inserted into a pipette (3 ml, Cat. No. 233-9525; Bio-Rad, Inc.) by first removing the pipette tip and then inserting the bead to a point 0.5 cm from the neck of the pipette. By
[41]
IMMUNOASSAY OF SULFATE-REDUCING BACTERIA
617
drawing the solution to be tested into the bulb of the pipette, and then expelling it, the pipette-bead assembly provides a convenient means of moving fluid through the bead. This format maximizes contact between the capture antibody on the surface of the porous bead and the solution to be tested.
Assay Buffers Samples to be assayed in the pipette-capture bead device should be in test buffer, which consists of Tris (50 mM, pH 7.5), sodium chloride (75 mM). SL-18 detergent (Olin Chemical Co., Stamford, CN), BSA (0.1%, w/v), and azide (0.02%, w/v). After the sample-conjugate mixture in test buffer has been cycled through the pipette-bead device, excess conjugate is washed out of the device with wash fluid [Tris (50 mM, pH 7.4) and azide (0.05%, w/v)]. The pH of the solution is adjusted to pH 7.4. For visualizing the amount of conjugate bound, a chromogenic substrate reagent consisting of 2.3 mM 5-bromo-4-chloroindolyl phosphate (Sigma Chemical Co.) in a 10% (v/v) 2-amino-2-methyl-l-propanol buffer at pH 10.2 containing 0.01% (w/v) MgCI2 (Sigma Chemical Co.) is drawn into the pipette-bead device.
Diatomaceous Earth Sample Processing Step This step relies on diatomaceous earth (DE) or a similar cell-entrapping matrix to facilitate collecting and washing the bacteria free of interfering solutes. In this process 10 ml of sample fluid is added to a sample collection vial containing a small amount of diatomaceous earth filtration medium (40-80 mg/ml sample). A filtration cap, containing a porous plastic frit (average pore size <10/zm) is then attached. The mixture is suspended by shaking the vial. The vial is then inverted and the suspended solids are allowed to settle into the filtration cap. The bottle is then squeezed to expel sample liquid to waste. During filtration, bacteria are entrapped in the filtration matrix and collected in the filtration cap. The bacteria are washed by reattaching the cap to a bottle containing 3.0 ml of wash fluid and squeezing the wash bottle. Cell lysis is achieved by 60-sec sonication cycles using a Branson model 450 sonifier equipped with a 1-mm probe tip. Celite (Celite Corp., Lompoc, CA) has been used successfully although effective cell recovery can be obtained with a number of different sorbent materials. Diatomaceous earths such as Kenite (200 or 700) (Witco Chemical Co., Chicago, IL), Celatom FW60 (Eagle Picher Co., Reno, NV), and Cuno M901 (AMF/Cuno Microfiltration Products, Meriden, CN) also give adequate recovery of sulfate-reducing bacteria.
618
[41]
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Test Performance
Specificity of Antibody Reagent Microtiter plate ELISA response of anti-D, desulfuricans G100A APS reductase crude antisera to crude extracts from sulfate-reducing bacteria, sulfide-oxidizing bacteria, as well as to control bacteria (which should not contain the enzyme) is shown in Table II. There appear to be three group-
T A B L E II CROSS-REACTIVITIES OF CRUDE EXTRACTS OF SULFATE-REDUCING BACTERIA AND SULFIDEOXIDIZING BACTERIA WITH ANTI-ADENOSINE-5 % PHOSPHOSULFATE REDUCTASE ANTIBODIES a
Organism
Strain G100A E L I S A b r e s p o n s e (%)
Desulfovibrio desulfuricans G100A A T C C 27774 API A T C C 13541
Desulfomicrobium baculatum Desulfovibrio vulgaris Desulfovibrio gigas Desulfovibrio salexigens Desulfovibrio multispirans
100 92 _+ 16 100 _+ 15 84 _+ 10 33 _+ 12 63 _+ 10 92 _+ I 1 76 _+ 9 69 _+ 15
Desulfotomaculum orientis Desulfotomaculum ruminis Desulfotomaculum nigrificans Desulfobulbus propion&us Desulfosarcina variabilis
48_+6 12_+3 22_+4 26_+6 21_+4
Thiobacillus denitrificans Chromatium oinosum Chlorobium thiosulfatophilum Escherichia coli Streptomyces lividans
8 +2 4 -+ 2 0 0 3 -+ 3
C o u r t e s y American Society for Microbiology. b The E L I S A r e s p o n s e at an optical density o f 410 n m as an average percentage of the Desulfovibrio desulfuricans G I 0 0 A r e s p o n s e over a range of from 6 to 600 ng o f crude extract protein per well for each organism.
[41]
IMMUNOASSAY OF SULFATE-REDUCING BACTERIA
619
ings of reactions, the most strongly cross-reactive (33-100% of G100A response) being mostly Desulfovibrio spp., including marine and freshwater strains. The second grouping consists of significantly less crossreactive (12-48% of G100A response) Desulfotomaculum, Desulfosarcina, and Desulfobulbus species. Cross-reactivity with Desulfotomaculum orientis is surprisingly high in light of the response with the other Desulfotomaculum species investigated. The third grouping consists of bacteria that lack APS reductase (E. coli and S. lividans), photosynthetic sulfide oxidizers (Chl. thiosulfatophilum and C. vinosum), and T. denitrificans. Of the non-sulfate-reducing bacteria investigated, the strongest response was consistently observed with T. denitrificans. It is anticipated from these results that antibody specificity will allow reasonable order-of-magnitude estimations of many Desulfovibrio spp., based on calibration of the response with cell suspensions of D. desulfuricans G100A, but will significantly underestimate the distantly related Desulfosarcina, Desulfobulbus, and Desulfotomaculum spp. A remedy for this would be the inclusion of antibodies to one or more of these organisms into the antibody capture and detection reagents. To see if these differences were actually due to the antigenic variation in the enzymes or to some other factor such as cellular enzyme content, enzyme extractability, or enzyme stability, enzymes from D. desulfuricans API. D. desulfuricans 27774, and Desulfomicrobium baculatum, D. vulgaris, Desulfotomaculum orientis, and T. denitrificans were purified to comparable specific activity and assayed by standard ELISA microtiter plate methodology (Table III). The data show that the purified enzyme reactivities paralleled crude cell lysate reactivities with the exception of Desulfotomaculum orientis, which gave essentially 100% of D. desulfuricans G100A response, suggesting that variations in response are due to antigenic differences in the enzymes themselves.
Sample Pretreatment Environmental samples may contain high concentrations of solid sediments, colloids, emulsions, soluble and insoluble salts, and industrial chemicals that can prevent or invalidate the immunoassay. To remove these potential interferences, sample processing is required prior to immunoassay. This is useful both to concentrate the small numbers of organisms found in field sample materials and to remove interfering materials that prevent analysis or cause inaccurate test results. Generally, sample treatment processes can involve centrifugation, membrane filtration or chemical precipitation; however, these procedures require laboratory facilities
620
[41]
SPECIAL TECHNIQUES TABLE III SPECIFIC ACTIVITIES AND CROsS-REACTIVITIES OF ENZYMES TO ANTI-ADENOSINE-St-PHosPHOSULFATEREDUCTASE POLYCLONAL ANTIBODIESa Strain G100A ELISA response (%)b
activityC
Desulfomicrobium baculatum
100 84 57 38
3.5 2.0 2.0 0.8
Desulfovibrio vulgaris Desulfotomaculum orientis Thiobacillus denitrificans
76 100 8
1.8 3.0 2.3
Organism
Specific
Desulfovibrio desulfuricans G100A API ATCC 27774
Courtesy American Society for Microbiology. b Purified protein, 240 ng per well. c Micromoles of ferricyanide reduced per minute per milligram of protein.
and are not readily accomplished in the field. We have found that bacteria can be efficiently entrapped in and recovered from small filters containing a diatomaceous earth (DE) as the entrapping matrix. Microorganisms can be effectively recovered over a wide range of cell concentrations typically encountered in environmental samples as shown in Table IV. In this example, production water from an oil well (courtesy of Conoco, Inc.) is seeded with varying concentrations ofD. desulfuricans TABLE IV SAMPLE PRETREATMENT CELL RECOVERY EFFICIENCY VERSUS SAMPLE CELL DENSITY OF Desulfooibrio desalfuricans GI00A Response (OD410) Cells/ml (added) 0.5 0.5 0.5 0.5 0
× x x x
107 106 105 104
Tris buffer ~
DE b
Filtration c
1.70 0.80 0.16 0.05 0.04
1.80 0.80 0.20 0.08 0.07
1.60 0.70 0.12 0.04 0.08
Control, no treatment. b Diatomaceous earth (Celite) at 80 mg/ml sample fluid. c Membrane filtration.
a
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SPECIALTECHNIQUES
[41]
using a Petroff-Hauser cell counter such that dilutions up to 108 cells/ml are available for testing. Figure 3 shows the field immunoassay response to standard cell dilutions of D. desulfuricans G100A. The following steps and incubation times were employed. 1. Cell suspensions (10 ml) are collected by diatomaceous earth pretreatment and washed free of residual medium with wash fluid. 2. The diatomaceous earth-bacteria mixture is then mixed with 1.5 ml of test buffer containing conjugate and sonicated for 30 sec. The soluble APS reductase-conjugate complex generated in this step is removed from the diatomaceous earth matrix by filtration. 3. The filtrate from step 2 is passed through the antibody-coated microporous bead-pipette device four times. The bead-pipette device is rinsed four times with wash fluid. 4. For the purpose of colorimetric quantitation, the beads are then removed from the pipette and placed in individual microtiter plate wells containing p-nitrophenol phosphate at 1 mg/ml in Sigma 221 buffer and allowed to incubate for 15 min before quantitation of the color formed. In an actual field assay a solution of 5-bromo-4-chloroindolyl phosphate is drawn directly into the bead-pipette device and cell density is estimated visually by comparison of bead color intensity with a calibrated color
2.0-
1.5. E eo
1.o0 0.5.
o . o
.
0
1
.
.
.
.
.
.
.
.
.
.
2 3 4 5 LOG Cell Density
.
6
7
FIG. 3. Response of field immunoassay assay to different concentrations of D. desulfuricans G100A. Response was numerically quantitated by removal of the capture bead from the pipette for color development in a microtiter plate well. (Courtesy National Association of Corrosion Engineers.)
[41]
IMMUNOASSAY OF SULFATE-REDUCING BACTERIA
623
chart relating color intensity to cell density of D. desulfuricans G100A. The curve in Fig. 3 relates cell numbers to optical density at 410 nm; note that the limit of detection is close to 10 4 cells/ml. To determine the extent of underestimation of poorly cross-reactive strains as well as cross-reactivity with a strong responding strain, D. desulfuricans API (100% o f D . desulfuricans G100A response) and Desulfomicrobium baculatum ( - 3 0 - 4 0 % of D. desulfuricans G100A response) are tested to check the correlation between actual cell counts and those determined by comparison with the colorimetric response by D. desulfuricans G100A. Field immunoassays were performed as described above for the D. desulfuricans G100A strain. Table V shows almost a 100-fold underestimation for cell numbers for the Desulfomicrobium baculatum strain even though the crude extract and purified enzyme ELISA data indicated only a 2- to 3-fold difference in response under equilibrium conditions. There is generally much less than an order of magnitude difference in cell numbers for the D. desulfuricans API strain. More importantly, the detection limit appears to be between 10 3 and 104 for D. desulfuricans API and 104 and 105 for Desulfomicrobium baculatum. It is important to emphasize that the field immunoassay is a short time course nonequilibrium assay as opposed to the microtiter plate assay, which is essentially TABLE V RESPONSE OF FIELD TEST FORMAT TO CELLS OF Desulfovibrio desulfuricans API AND Desulfomicrobium baculatum AS DETERMINED BY COLORIMETRIC RESPONSE RELATIVE TO
Desulfovibrio desulfuricans GI00A Cell density by immunoassay b Actual cell density a
D. desulfuricans API
0
10z 103 104 105 106 107 108
2 2 9 2 5
0 0 0 × 103 x 105 x 106 × 107 X 107
Dsm. baculatum
6 6 4 9 4
0 0 0 × 102 x 102 × 104 × 104 × 106
Cell density as determined by microscopic counts using a PetroffHauser counter. b Cell density as determined by rapid immunoassay and comparison of color response with D.desulfuricans G100A standard response curve.
624
SPECIALTECHNIQUES
[41]
an equilibrium assay. The nonequilibrium format apepars to be more sensitive to differences in affinity between antibody and the different APS reductases. However, we have found, using the identical reagents in the microtiter plate format, that the detection limit for D. desulfuricans G 100A is 105 to 106 cells/ml after a total incubation time of 2 hr as opposed to 2-3 min for the capture-bead format. Concluding Remarks We describe a method for estimating cell numbers of sulfate-reducing bacteria in crude environmental samples. There are two unique and key elements to this method: (1) the sample pretreatment step, which isolates the bacteria and removes soluble interferences, and (2) the antibodycoated microporous capture-bead device, which enables rapid and efficient capture of the antigen from the sample. At present antibody specificity restricts the sensitivity of the test to Desulfovibrio spp., however, inclusion of antibodies to APS reductases from other genera into the antibody reagent should expand the overall specificity of the test.