- Email: [email protected]
[21] Solid-phase radioimmunoassay for bacterial lipopolysaccharide
264
TOXINS
[21]
they left the phenyl-Sepharose column. The high-molecular-weight fractions were straw-colored due to the presence of food components so that elution from Sephadex G-25 could also be followed visually. The fractions were pooled and 100/zl duplicates were used for the assay of SEC2. Recoveries, calculated from the standard curve, were about 70% (Table III), which was sufficient to enable the detection of as little as 0.14 ng SEC_~/ml in 35 ml of the extract. This level of SEC2 could not be detected directly by the radioimmunoassay as it is currently performed. The added sensitivity accrues from an approximate 10-fold concentration of the toxin. The technique is applicable to other staphylococcal enterotoxins such as types A, B, and E. For example, with enterotoxin A we detected as little as 0.2 ng/g of naturally contaminated food (unpublished results).
[2 1] S o l i d - P h a s e By
Radioimmunoassay Lipopolysaccharide
for Bacterial
D I A N E M . JACOBS a n d JAN A . GUTOWSKI
Lipopolysaccharide (LPS) is the endotoxic constituent of gram-negative bacteria. Present in up to 20-30% it, together with various lipids and proteins, makes up the outer membrane of the cell. Gram-negative bacteria when allowed to invade and multiply in an animal host release LPS into the circulatory system which in turn can give rise to a multitude of pathophysiological manifestations such as fever, shock and even death.X A number of biological, 2-4 chemical, 5,6 and enzymatic r assays are at present available for estimating endotoxin levels. However, these tend to be semiquantitative at best and interpretable only when applied under specific and restrictive test conditions. The development of a radioimmunoassay for lipopolysaccharide has in the past been hindered by the unique chemical and physical characteris-
i S. Kadis, G. Weinbaum, and S. J. Ajl (eds.), "Microbial Toxins," Vol. V. Academic Press, New York, 1971. 2 K. C. Milner and R. A. Finkelstein, J. Infect. Dis. 116, 529 (1966). 3 W. R. Keene, H. R. Silberman, and M. Landy, J. Clin. Invest. 40, 295 (1961). 4 R. E. Pierone, E. J. Broderick, A. Bundeally, and L. Levine, Proc. Soc. Exp. Biol. Med. 133, 790 (1970). 5 V. S. Waravdekar and L. D. Saslow, J. Biol. Chem. 234, 1945 (1959). s y . D. Karkhanis, J. Y. Zeltner, J. J. Jackson, and D. J. Carlo, Anal. Biochem. 85, 595 (1978). 7 R. Nandan and D. R. Brown, £. Lab. Clin. Med. 89, 910 (1977).
METHODS IN ENZYMOLOGY, VOL. 84
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181984-1
[21]
SOLID-PHASE RIA FOR BACTERIAL LPS
265
tics of the molecule. Thus the absence of residues which lend themselves to iodination by conventional labeling techniques has necessitated the attachment of various tyrosine-like ligands; the difficulty in raising an antiserum of sufficiently high titer has led to the establishment of an immunization schedule specific for this molecule; and its high nonspecific adherence to precipitated protein has prompted the development of a solid-phase assay system. The technique of radioimmunoassay provides a sensitive and quantitative method of measuring lipopolysaccharide unaffected by various inhibitors or cross-reactive molecules afflicting other presently available assays. Here we present a radioimmunoassay for E. coli 055:B5 lipopolysaccharide.8 No data will be given as to the cross-reactivity of 055 : B5 antiserum in detecting other strains of LPS. Principle The radioimmunoassay for lipopolysaccharide is based on the competition of binding to anti-LPS IgG coated polystyrene tubes of 125I-labeled and native LPS. After incubation, bound and free LPS are separated by aspiration and the tubes are counted after being washed with saline. Thiobarbituric Acid (TBA) Determination of L P S The thiobarbituric acid method 5 is used for quantitating LPS and derivatized LPS in column fractions and pooled solutions using purified E. coli 055:B5 LPS as standard. It has been modified by Morrison and Leive, 9 and is used in our laboratory as described below. Reagents 2-Thiobarbituric acid, pH 2.0. Mix 0.71 g crystalline thiobarbituric acid in 90 ml H20 and add 0.7 ml 1.0 N NaOH. Shake in warm water until solution is clear. Adjust to 100 ml and filter. Solution is said to be stable for 30 days, but is usually made fresh weekly. Dilute 1 : 2 in water just before use Periodic acid reagent: 0.025 N sodium periodate in 0.125 N H2SO4 Arsenite solution: 2% reagent grade sodium arsenite in 0.5 N HC1 0.2 N H~SO4 10 N NaOH Procedure. Add 0.2 ml sample (1-100/xg LPS) to 0.1 ml 0 . 2 N H2SO4. Heat in boiling water bath 15 rain using condensers (marbles) on s j. A. Gutowski and D. M. Jacobs, Immunol. Commun. 8, 347 (1979). D. C. Morrison and L. Leiv¢, J. Biol. Chem. 250, 2911 (1975).
266
TOXINS
[21]
tubes. Add 0.2 ml periodic acid reagent and heat at 55° for 22 min. Add 0.4 ml arsenite solution and mix immediately. Add 1.6 ml thiobarbituric acid. Heat 12 min in boiling water bath. Allow to cool. Read at 532 nm. If a precipitate has formed in any tubes, add 2 drops NaOH to all tubes before reading. Isolation of L P S This phenol-water extraction procedure was first described by Westphal and Jann 1° and subsequently modified by Morrison and Leive. 9 The latter modifications have been adopted and used in our laboratory for extracting E. coil 055 : B5 as follows.
Reagents Bacteria: 50 g wet weight in 50 ml saline or 12.5 g dry weight suspended in 50 ml saline (lyophilized bacteria obtained from the New England Enzyme Center, Boston, MA.) 90% Phenol (Mallinckrodt). Place 45 g phenol in 100 ml graduated cylinder. Carefully add 4 ml boiling distilled water. Warm to 56°. Adjust to 50 ml. Heat to 68° in water bath Sepharose 4B (Pharmacia) column, 0.1 M Tris buffer, pH 8.0 with 0.05% azide 1 M MgCI~ Ribonuclease A (5 x crystallized, Type l-A, Sigma) Pronase (protease from Streptomyces griseus, repurified, Type VI, Sigma) Procedure. Rapidly add equal volume 90% phenol to bacterial suspension and mix well at 68° for 30 min. Transfer suspension to 25-ml screwcapped Corex tubes and centrifuge at 10,000 rpm for 30 min at 4 °. Remove and reserve top aqueous phase. Wash pellet and phenol phase with 50 ml saline, and centrifuge as before. Remove aqueous phase and combine with aqueous phase from first extraction. Discard phenol phase which is very dark and has large, sticky pellet. Dialyze aqueous phase against several changes of several liters of saline until no odor of phenol is discernible. Add 0.5 ml 1 M MgCl~ for each 200 ml. Add RNase to 20 /zg/ml, and incubate 1 hr at 37°. Add Pronase to 20/xg/ml, incubating overnight at 37°. Concentrate to 25 ml by Amicon ultra.filtration using PM30 filter. Load preparation onto 3-liter Sepharose 4B column. Diameter/height ratio is not crucialmlaboratory column is 10 x 40 cm. Elute with 0.1 M 10 O. Westphal and K. Jann, in "Methods in Carbohydrate Chemistry" (R. L. Whistler, ed.), p. 83. Academic Press, New York, 1965.
[21]
SOLID-PHASE RIA FOR BACTERIAL LPS
267
Tris buffer, pH 8.0 containing 0.05% azide, collecting 15-20 ml fractions per tube. Read fractions at 260 nm to detect nucleic acids. Assay 20/.d alternate fractions with TBA for LPS. Peaks should overlap only minimally. Pool and concentrate LPS peak and dialyze against pyrogen-free water. Preparation of IgG Fraction of Antiserum
Immunization and Preparation of Serum. Bacteria are grown in minireal medium for 18 hr and heated at 100° for 2.5 hr. For immunization, the suspension is adjusted with saline to ODe00 nm= 0.7, approximately 109 organisms per milliter. New Zealand white rabbits are injected intravenously with 1 ml suspension three times per week for at least 4 months. If dried bacteria are available, 0.2 ml of a suspension of 1 mg/ml in saline can be used. Rabbits are allowed to rest for 10 days before bleeding. (A test bleed is recommended.) Blood is collected, allowed to clot at room temperature, and held at 4 ° overnight. Serum is separated from the clot after centrifugation. Chronic immunization is necessary to induce high titers of IgG anti-LPS antibody. High titered antiserum will give good precipitation lines when tested by immunodiffusion. Ouchterlony plates made up of 1.5% Ionagar No. 2 in barbital acetate buffer, pH 8.6 (5.4 g sodium barbital, 4.3 g sodium acetate trihydrate, and 58.2 ml 0.1 N HC1 per liter) containing 0.5 M glycine are used. Wells are filled with undiluted antiserum and LPS samples at 2 mg/ml. Line can be seen after incubation overnight at room temperature. Fractionation of Serum. The IgG fraction of serum is prepared by chromatography on DEAE Afli-Gel Blue (Bio-Rad Laboratories) using the directions supplied with the resin. In our laboratory we separate 3 ml serum on a 21 ml column equilibrated with a buffer composed of 0.02 M Tris-HCl, 0.011 M NaC1, pH 7.2. The serum is dialyzed against the buffer before application to the column and eluted with three bed volumes of the starting buffer. Fractions are monitored at 280 nm and peak fractions pooled for use. Protein concentration of the pool is determined by any routine laboratory procedure. Use of this resin produced IgG which gave more reproducible sigmoid curves in the final RIA than IgG prepared on regular DEAE. Preparation of Derivatized L P S
Reagents p-Hydroxyphenylacetic acid (pHPAA) (Aldrich Chemical Co.) 4-Hydroxyphenethylamine (Tyramine, T) (Sigma Chemical Co.)
268
TOXINS
[21]
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl (EDC) (Pierce Chemical Co.) Glutaraldehyde, 50% (Fisher Scientific Co.) Sephadex G-75, Sepharose 4B (Pharmacia Fine Chemicals) 0.01 N NaOH Sodium phosphate buffer: 0.1 M, pH 6.5 Sodium acetate buffer: 0.5 M, pH 5.0 Glycine: 2 M, pH 8.0 Ethanol
Procedure p-Hydroxyphenylacetic Acid-LPS (pHPAA-LPS). 11,12To a solution of pHPAA (11.4 mg) in 1.25 ml of water is added 6 mg EDC. The pH is adjusted to 5 - 6 with 0.01 N NaOH, and the mixture is incubated at 37° for 8 hr in the dark with stirring. LPS (3 mg) in 40/zl of water is now added and the incubation is continued in the dark at 37° for a further 24 hr with stirring, maintaining pH 5 - 6 with 0.01 N NaOH. The pHPAA-LPS is purified on a Sephadex G-75 column (1 x 20 cm) equilibrated in water, collecting 1-ml fractions. The desired material is eluted in the first of two peaks (fractions 7-14) and pooled. Yield is ca. 2.5 mg. Tyramine-LPS (T-LPS). 13 To a pale yellow solution of tyramine (16.4 mg), dissolved with warming in 3 ml of ethanol, is added 2 ml of sodium phosphate buffer (0.1 M, pH 6.5), 3 mg of LPS, and 27 mg of glutaraldehyde. The mixture is agitated gently for 3 hr at room temperature at which time the reaction is terminated by addition of 200 ttl of sodium acetate buffer (0.5 M, pH 5.0) over 30 sec. Unreacted aldehyde groups are blocked by addition of 2.0 ml of 2 M glycine, pH 8.0, and mixing is continued for 2 hr at room temperature. A slight precipitate which occasionally forms during the reaction is removed by centrifugation and the derivatized LPS is purified on a column (1 × 50 cm) of Sepharose 4B equilibrated in water, collecting 1.3-ml fractions. The derivatized LPS is eluted in the first of two peaks (fractions 5-11) and pooled. Yield is about 2.0 mg. Both pHPAA-LPS and T-LPS are indistinguishable from the underivatized endotoxin by various biophysical, immunological, and biological criteria. 8
~1 j. C. Sheehan and G. P. Hess, J. Am. Chem. Soc. 77, 1067 (1955). 1~ H. G. Khorana, Chem. Ind. (London) 1087 (1955). ~3 D. J. Ford, R. Radin, and A. J. Pesce, Immunochemistry 15, 237 (1978).
[21]
SOLID-PHASE RIA FOR BACTERIAL LPS
Iodination of Derivatized
269
L P S 14
Reagents
NaX25I: /> 17 mCi/~g, carrier-free IMS-30 (Amersham/Searle) Phosphate buffers: 0.1 and 0.5 M sodium phosphate, pH 7.2 Chloramine-T: 1 mg/ml in 0.1 M phosphate buffer (Eastman-Kodak) Sodium metabisulphite: 2 mg/ml in 0.1 M phosphate buffer (Fisher Scientific Co.) Borate buffer: prepared by adjusting pyrogen free saline (0.9% NaCI, Travenol Laboratories) to 50 mM in sodium borate, 50 mM in CaC12, and 0.02% NAN3, pH 8.0 Derivatized LPS (T-LPS or pHPAA-LPS), 500/~g/ml in 0.1 M phosphate buffer Sephadex G-75 (Pharmacia Fine Chemicals) For transfer of low concentrations of LPS glass equipment must be used throughout. Procedure. Forty microliters 0.5 M phosphate buffer, and 10/~g derivatized LPS are added to the vial containing 1 mCi Na125I and mixed by vortexing. The mixture is then treated with 20/~1 of chloramine-T over 30 sec with vigorous vortexing. The reaction is stopped with 50/zl sodium metabisulfite followed by 100/~1 of borate buffer and vortexed. The 125I-LPS is purified on a column (10 ml glass pipette) of Sephadex G-75 equilibrated in borate buffer and 1-ml fractions collected. The purified material is eluted in fractions 6 - 7 (Peak I) and free iodide in fractions 10-11 (Peak II). It is stored at 4 ° until use. Normally, about 10% of recovered 125Iis incorporated into the derivatized endotoxin which has a final minimum specific activity of ca. 2 - 5 mCi/mg based on a total recovery of column applied material. As it is not possible to accurately measure this small amount of LPS in the eluate by the TBA assay, the calculated specific activity is only a minimum estimate. We have, however, used a more direct approach to determine the specific activity by increasing the amount of LPS used in the labeling procedure. When a column is loaded with 170/zg pHPAA-LPS and the eluted fractions assayed by the TBA method, 8-10% of the starting material is recovered in one peak corresponding to Peak I. When the same amount of derivatized LPS is iodinated and fractionated, Peak I contains 8-10% of the starting material and Peak II, 26-30% of the starting material. Peak I therefore contains 21-33% of recovered radioactivity. (Data based on routine iodinations of this type carried out over a 2-year period.) 14F. C. Greenwood, W. M. Hunter, and J. S. Glover, Biochem. J. 89, 114 (1963).
270
TOXINS
[21]
These data suggest that (1) more iodide is incorporated into LPS when a larger amount is labeled and (2) 90% of the applied LPS sticks to the column nonspecifically, whether or not it is iodinated. On the assumption that unrecovered radioactivity is all in column-adherent LPS, we can estimate the amount of LPS recovered in Peak I and calculate the specific activity which is 2 - 5 mCi/mg. If the same fraction of LPS is recovered when 10 fig is iodinated, the actual specific activity could well be an order of magnitude higher.
Radioimmunoassay
Reagents 12 x 75 mm polystyrene tubes (Falcon No. 2052) Borate-buffered saline (BBS) with azide: prepared by adjusting pyrogen-free saline (0.9% NaC1, Travenol Laboratories) to 50 mM in borate and 0.02% NaNa at pH 8.0 ( " p H 8.0 BBS") and pH 9.5 ( " p H 9.5 BBS") Bovine serum albumin (BSA): 0.5% in pH 8.0 BBS (Sigma, RIA grade) IgG fraction of rabbit anti-LPS diluted in pH 9.5 BBS 37° shaker/water bath or a shaker (R-2 reciprocator, New Brunswick Scientific Co., set at 200 strokes/rain) in a 37° warm room
Procedure Coating of Tubes with Antibody. One-haft milliter of an appropriate dilution (see next section) of the IgG fraction of rabbit anti-LPS made up in pH 9.5 BBS is carefully pipetted into the bottom of each polystyrene tube. After 1 hr at room temperature the antibody solution is removed by aspiration and pooled for reuse. The tubes are incubated with 1 ml of 0.5% BSA for a further 30 rain at room temperature. After the BSA is discarded, the tubes are washed twice with 3 ml o f p H 8.0 BBS and drained inverted over absorbent tissue at 4°. Normally the tubes are used within a few hours, but they will maintain their binding capacity for 24-36 hr when stored at 4 °. The pooled antiserum has a shelf life of at least 1 month when kept at 4 °, but should be regularly monitored for 100% binding e ~ ciency if used repeatedly. Contamination with extraneous endotoxin is usually undetectable. Coating with 0.5% BSA does not normally improve the assay. Nonetheless, this procedure is recommended to preclude any unexpected nonspecific binding of I~SI-LPS to tube surfaces. Likewise, increasing the tube-coating time from 1 hr up to 4 hr does not appreciably increase the amount of IgG bound.
[21]
SOLID-PHASE RIA FOR BACTERIAL LPS
271
Determination of Antibody Titer. Anti-LPS IgG fraction is serially diluted in pH 9.5 BBS in 1 ml volumes in glass tubes. Polystyrene tubes are coated with 0.5 ml each dilution as described above. Coated tubes are subsequently incubated with ca. 15,000 cpm 125I-derivatized LPS in 600/zl pH 8.0 BBS and incubated with shaking for 4 hr at 37°. The solutions are removed by aspiration, the tubes are washed once with 3 ml of saline, and bound radioactivity determined in a gamma scintillation spectrometer. From a plot of percentage of added radioactivity bound against log10 concentration of anti-LPS IgG the antibody titer is estimated as the concentration of IgG which results in a 50% binding of tracer relative to the maximum observed binding. This IgG concentration is used in coating RIA tubes for detection of LPS. Detection ofLPS. LPS samples are serially diluted in pH 8.0 BBS in glass tubes. Four hundred microliters of each sample and 100/zl pH 8.0 BBS are incubated overnight at 4 ° in antibody-coated polystyrene tubes on a reciprocating shaker. One hundred microliters of 125I-derivatized LPS (ca. 15,000 cpm) are added, and the tubes are incubated for a further 2 hr at 37°. The reaction mixture is then removed by aspiration, the tubes are washed once with 3 ml of saline, and counted. Two controls are run concurrently: Bo (100% binding) contains 500/~1 of pH 8.0 BBS and 100/zl of ~25I-LPS; NSB (nonspecific binding) contains the same addition as B0 but uses tubes coated with 0.5% BSA only and no antibody. Samples and controls are run at least in duplicate, preferably triplicate. 10(3
60
20 •
~-I
0.0t
@
•
I
I-
.I.
.I
0.t
t.0
t0
100
~g/ml LPS FIG. 1. Detection of LPS in radioimmunoassay using IgG prepared on Affi-Gel Blue dextran.
272
TOXINS
[22]
Percentage bound is calculated from the relationship: Sample cpm - NSB cpm x 100% B0 cpm Standard curves are plotted as percentage tracer bound against log10 LPS concentration in/zg/ml (Fig. 1). LPS content of unknown is estimated from the dilution factor at 50% binding relative to that of the standard sample. In the case ofE. coli 055: B5 LPS, a sensitivity range of approximately 10-500 ng/ml was obtained) Nonspecific binding and 100% binding were about 5 and 60%, respectively, of the total counts added.
[22] R a d i o i m m u n o a s s a y s o f T h y r o x i n e (T4), 3 , 5 , 3 ' - T r i i o d o t h y r o n i n e (Ta) , 3 , 3 ' , 5 ' - T r i i o d o t h y r o n i n e ( R e v e r s e T 3 , rTa), a n d 3 , 3 ' - D i i o d o t h y r o n i n e (Tz) By W I L M A R M . W I E R S I N G A a n d I N D E R J . C H O P R A
Introduction Although there has been a decided improvement in our understanding of the field of endocrinology during the last decade because of the many rapid developments, the resulting increase in complexity of certain issues has led to confusion in some instances. For example, we now know that substances synthesized within an endocrine cell are not necessarily those conventionally viewed as the hormone, but merely are precursors (e.g., preproparathormone and proparathormone in case of parathormone). Along the same line, the substance secreted by an endocrine cell into the bloodstream is not necessarily the one that ultimately provokes the hormonal effect in the target tissues but only a prehormone (e.g., testosterone for dihydrotestosterone). The hormonal effects in the target cells may be modulated not just by circulating hormone levels but also by the receptor state (e.g., number and affinity of receptors for the hormone), and in some cases also by postreceptor alterations. Recent discoveries involving thyroxine (I"4) provide an excellent illustration of these complexities, First, T4 synthesized in the thyroid gland is secreted unchanged but is also converted intrathyroidally to a more active triiodothyronine (Ta). x Second, T4 released into circulation is monodeiodinated in the extrathyroidal tissues into either the more potent hormone i H. Ishii, K. Tanaka, K. Naito, M. Nishikawa, and M. Inada, Annu. Meet., 62nd, Endocr. Soc., Washington, D.C.p. 202, Abstr. 512 (1980).
METHODS IN ENZYMOLOGY, VOL. 84
Copyright© 1982by AcademicPress, Inc. All rightsof reproductionin any form reserved. ISBN 0-12-181984-1
TOXINS
[21]
they left the phenyl-Sepharose column. The high-molecular-weight fractions were straw-colored due to the presence of food components so that elution from Sephadex G-25 could also be followed visually. The fractions were pooled and 100/zl duplicates were used for the assay of SEC2. Recoveries, calculated from the standard curve, were about 70% (Table III), which was sufficient to enable the detection of as little as 0.14 ng SEC_~/ml in 35 ml of the extract. This level of SEC2 could not be detected directly by the radioimmunoassay as it is currently performed. The added sensitivity accrues from an approximate 10-fold concentration of the toxin. The technique is applicable to other staphylococcal enterotoxins such as types A, B, and E. For example, with enterotoxin A we detected as little as 0.2 ng/g of naturally contaminated food (unpublished results).
[2 1] S o l i d - P h a s e By
Radioimmunoassay Lipopolysaccharide
for Bacterial
D I A N E M . JACOBS a n d JAN A . GUTOWSKI
Lipopolysaccharide (LPS) is the endotoxic constituent of gram-negative bacteria. Present in up to 20-30% it, together with various lipids and proteins, makes up the outer membrane of the cell. Gram-negative bacteria when allowed to invade and multiply in an animal host release LPS into the circulatory system which in turn can give rise to a multitude of pathophysiological manifestations such as fever, shock and even death.X A number of biological, 2-4 chemical, 5,6 and enzymatic r assays are at present available for estimating endotoxin levels. However, these tend to be semiquantitative at best and interpretable only when applied under specific and restrictive test conditions. The development of a radioimmunoassay for lipopolysaccharide has in the past been hindered by the unique chemical and physical characteris-
i S. Kadis, G. Weinbaum, and S. J. Ajl (eds.), "Microbial Toxins," Vol. V. Academic Press, New York, 1971. 2 K. C. Milner and R. A. Finkelstein, J. Infect. Dis. 116, 529 (1966). 3 W. R. Keene, H. R. Silberman, and M. Landy, J. Clin. Invest. 40, 295 (1961). 4 R. E. Pierone, E. J. Broderick, A. Bundeally, and L. Levine, Proc. Soc. Exp. Biol. Med. 133, 790 (1970). 5 V. S. Waravdekar and L. D. Saslow, J. Biol. Chem. 234, 1945 (1959). s y . D. Karkhanis, J. Y. Zeltner, J. J. Jackson, and D. J. Carlo, Anal. Biochem. 85, 595 (1978). 7 R. Nandan and D. R. Brown, £. Lab. Clin. Med. 89, 910 (1977).
METHODS IN ENZYMOLOGY, VOL. 84
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181984-1
[21]
SOLID-PHASE RIA FOR BACTERIAL LPS
265
tics of the molecule. Thus the absence of residues which lend themselves to iodination by conventional labeling techniques has necessitated the attachment of various tyrosine-like ligands; the difficulty in raising an antiserum of sufficiently high titer has led to the establishment of an immunization schedule specific for this molecule; and its high nonspecific adherence to precipitated protein has prompted the development of a solid-phase assay system. The technique of radioimmunoassay provides a sensitive and quantitative method of measuring lipopolysaccharide unaffected by various inhibitors or cross-reactive molecules afflicting other presently available assays. Here we present a radioimmunoassay for E. coli 055:B5 lipopolysaccharide.8 No data will be given as to the cross-reactivity of 055 : B5 antiserum in detecting other strains of LPS. Principle The radioimmunoassay for lipopolysaccharide is based on the competition of binding to anti-LPS IgG coated polystyrene tubes of 125I-labeled and native LPS. After incubation, bound and free LPS are separated by aspiration and the tubes are counted after being washed with saline. Thiobarbituric Acid (TBA) Determination of L P S The thiobarbituric acid method 5 is used for quantitating LPS and derivatized LPS in column fractions and pooled solutions using purified E. coli 055:B5 LPS as standard. It has been modified by Morrison and Leive, 9 and is used in our laboratory as described below. Reagents 2-Thiobarbituric acid, pH 2.0. Mix 0.71 g crystalline thiobarbituric acid in 90 ml H20 and add 0.7 ml 1.0 N NaOH. Shake in warm water until solution is clear. Adjust to 100 ml and filter. Solution is said to be stable for 30 days, but is usually made fresh weekly. Dilute 1 : 2 in water just before use Periodic acid reagent: 0.025 N sodium periodate in 0.125 N H2SO4 Arsenite solution: 2% reagent grade sodium arsenite in 0.5 N HC1 0.2 N H~SO4 10 N NaOH Procedure. Add 0.2 ml sample (1-100/xg LPS) to 0.1 ml 0 . 2 N H2SO4. Heat in boiling water bath 15 rain using condensers (marbles) on s j. A. Gutowski and D. M. Jacobs, Immunol. Commun. 8, 347 (1979). D. C. Morrison and L. Leiv¢, J. Biol. Chem. 250, 2911 (1975).
266
TOXINS
[21]
tubes. Add 0.2 ml periodic acid reagent and heat at 55° for 22 min. Add 0.4 ml arsenite solution and mix immediately. Add 1.6 ml thiobarbituric acid. Heat 12 min in boiling water bath. Allow to cool. Read at 532 nm. If a precipitate has formed in any tubes, add 2 drops NaOH to all tubes before reading. Isolation of L P S This phenol-water extraction procedure was first described by Westphal and Jann 1° and subsequently modified by Morrison and Leive. 9 The latter modifications have been adopted and used in our laboratory for extracting E. coil 055 : B5 as follows.
Reagents Bacteria: 50 g wet weight in 50 ml saline or 12.5 g dry weight suspended in 50 ml saline (lyophilized bacteria obtained from the New England Enzyme Center, Boston, MA.) 90% Phenol (Mallinckrodt). Place 45 g phenol in 100 ml graduated cylinder. Carefully add 4 ml boiling distilled water. Warm to 56°. Adjust to 50 ml. Heat to 68° in water bath Sepharose 4B (Pharmacia) column, 0.1 M Tris buffer, pH 8.0 with 0.05% azide 1 M MgCI~ Ribonuclease A (5 x crystallized, Type l-A, Sigma) Pronase (protease from Streptomyces griseus, repurified, Type VI, Sigma) Procedure. Rapidly add equal volume 90% phenol to bacterial suspension and mix well at 68° for 30 min. Transfer suspension to 25-ml screwcapped Corex tubes and centrifuge at 10,000 rpm for 30 min at 4 °. Remove and reserve top aqueous phase. Wash pellet and phenol phase with 50 ml saline, and centrifuge as before. Remove aqueous phase and combine with aqueous phase from first extraction. Discard phenol phase which is very dark and has large, sticky pellet. Dialyze aqueous phase against several changes of several liters of saline until no odor of phenol is discernible. Add 0.5 ml 1 M MgCl~ for each 200 ml. Add RNase to 20 /zg/ml, and incubate 1 hr at 37°. Add Pronase to 20/xg/ml, incubating overnight at 37°. Concentrate to 25 ml by Amicon ultra.filtration using PM30 filter. Load preparation onto 3-liter Sepharose 4B column. Diameter/height ratio is not crucialmlaboratory column is 10 x 40 cm. Elute with 0.1 M 10 O. Westphal and K. Jann, in "Methods in Carbohydrate Chemistry" (R. L. Whistler, ed.), p. 83. Academic Press, New York, 1965.
[21]
SOLID-PHASE RIA FOR BACTERIAL LPS
267
Tris buffer, pH 8.0 containing 0.05% azide, collecting 15-20 ml fractions per tube. Read fractions at 260 nm to detect nucleic acids. Assay 20/.d alternate fractions with TBA for LPS. Peaks should overlap only minimally. Pool and concentrate LPS peak and dialyze against pyrogen-free water. Preparation of IgG Fraction of Antiserum
Immunization and Preparation of Serum. Bacteria are grown in minireal medium for 18 hr and heated at 100° for 2.5 hr. For immunization, the suspension is adjusted with saline to ODe00 nm= 0.7, approximately 109 organisms per milliter. New Zealand white rabbits are injected intravenously with 1 ml suspension three times per week for at least 4 months. If dried bacteria are available, 0.2 ml of a suspension of 1 mg/ml in saline can be used. Rabbits are allowed to rest for 10 days before bleeding. (A test bleed is recommended.) Blood is collected, allowed to clot at room temperature, and held at 4 ° overnight. Serum is separated from the clot after centrifugation. Chronic immunization is necessary to induce high titers of IgG anti-LPS antibody. High titered antiserum will give good precipitation lines when tested by immunodiffusion. Ouchterlony plates made up of 1.5% Ionagar No. 2 in barbital acetate buffer, pH 8.6 (5.4 g sodium barbital, 4.3 g sodium acetate trihydrate, and 58.2 ml 0.1 N HC1 per liter) containing 0.5 M glycine are used. Wells are filled with undiluted antiserum and LPS samples at 2 mg/ml. Line can be seen after incubation overnight at room temperature. Fractionation of Serum. The IgG fraction of serum is prepared by chromatography on DEAE Afli-Gel Blue (Bio-Rad Laboratories) using the directions supplied with the resin. In our laboratory we separate 3 ml serum on a 21 ml column equilibrated with a buffer composed of 0.02 M Tris-HCl, 0.011 M NaC1, pH 7.2. The serum is dialyzed against the buffer before application to the column and eluted with three bed volumes of the starting buffer. Fractions are monitored at 280 nm and peak fractions pooled for use. Protein concentration of the pool is determined by any routine laboratory procedure. Use of this resin produced IgG which gave more reproducible sigmoid curves in the final RIA than IgG prepared on regular DEAE. Preparation of Derivatized L P S
Reagents p-Hydroxyphenylacetic acid (pHPAA) (Aldrich Chemical Co.) 4-Hydroxyphenethylamine (Tyramine, T) (Sigma Chemical Co.)
268
TOXINS
[21]
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl (EDC) (Pierce Chemical Co.) Glutaraldehyde, 50% (Fisher Scientific Co.) Sephadex G-75, Sepharose 4B (Pharmacia Fine Chemicals) 0.01 N NaOH Sodium phosphate buffer: 0.1 M, pH 6.5 Sodium acetate buffer: 0.5 M, pH 5.0 Glycine: 2 M, pH 8.0 Ethanol
Procedure p-Hydroxyphenylacetic Acid-LPS (pHPAA-LPS). 11,12To a solution of pHPAA (11.4 mg) in 1.25 ml of water is added 6 mg EDC. The pH is adjusted to 5 - 6 with 0.01 N NaOH, and the mixture is incubated at 37° for 8 hr in the dark with stirring. LPS (3 mg) in 40/zl of water is now added and the incubation is continued in the dark at 37° for a further 24 hr with stirring, maintaining pH 5 - 6 with 0.01 N NaOH. The pHPAA-LPS is purified on a Sephadex G-75 column (1 x 20 cm) equilibrated in water, collecting 1-ml fractions. The desired material is eluted in the first of two peaks (fractions 7-14) and pooled. Yield is ca. 2.5 mg. Tyramine-LPS (T-LPS). 13 To a pale yellow solution of tyramine (16.4 mg), dissolved with warming in 3 ml of ethanol, is added 2 ml of sodium phosphate buffer (0.1 M, pH 6.5), 3 mg of LPS, and 27 mg of glutaraldehyde. The mixture is agitated gently for 3 hr at room temperature at which time the reaction is terminated by addition of 200 ttl of sodium acetate buffer (0.5 M, pH 5.0) over 30 sec. Unreacted aldehyde groups are blocked by addition of 2.0 ml of 2 M glycine, pH 8.0, and mixing is continued for 2 hr at room temperature. A slight precipitate which occasionally forms during the reaction is removed by centrifugation and the derivatized LPS is purified on a column (1 × 50 cm) of Sepharose 4B equilibrated in water, collecting 1.3-ml fractions. The derivatized LPS is eluted in the first of two peaks (fractions 5-11) and pooled. Yield is about 2.0 mg. Both pHPAA-LPS and T-LPS are indistinguishable from the underivatized endotoxin by various biophysical, immunological, and biological criteria. 8
~1 j. C. Sheehan and G. P. Hess, J. Am. Chem. Soc. 77, 1067 (1955). 1~ H. G. Khorana, Chem. Ind. (London) 1087 (1955). ~3 D. J. Ford, R. Radin, and A. J. Pesce, Immunochemistry 15, 237 (1978).
[21]
SOLID-PHASE RIA FOR BACTERIAL LPS
Iodination of Derivatized
269
L P S 14
Reagents
NaX25I: /> 17 mCi/~g, carrier-free IMS-30 (Amersham/Searle) Phosphate buffers: 0.1 and 0.5 M sodium phosphate, pH 7.2 Chloramine-T: 1 mg/ml in 0.1 M phosphate buffer (Eastman-Kodak) Sodium metabisulphite: 2 mg/ml in 0.1 M phosphate buffer (Fisher Scientific Co.) Borate buffer: prepared by adjusting pyrogen free saline (0.9% NaCI, Travenol Laboratories) to 50 mM in sodium borate, 50 mM in CaC12, and 0.02% NAN3, pH 8.0 Derivatized LPS (T-LPS or pHPAA-LPS), 500/~g/ml in 0.1 M phosphate buffer Sephadex G-75 (Pharmacia Fine Chemicals) For transfer of low concentrations of LPS glass equipment must be used throughout. Procedure. Forty microliters 0.5 M phosphate buffer, and 10/~g derivatized LPS are added to the vial containing 1 mCi Na125I and mixed by vortexing. The mixture is then treated with 20/~1 of chloramine-T over 30 sec with vigorous vortexing. The reaction is stopped with 50/zl sodium metabisulfite followed by 100/~1 of borate buffer and vortexed. The 125I-LPS is purified on a column (10 ml glass pipette) of Sephadex G-75 equilibrated in borate buffer and 1-ml fractions collected. The purified material is eluted in fractions 6 - 7 (Peak I) and free iodide in fractions 10-11 (Peak II). It is stored at 4 ° until use. Normally, about 10% of recovered 125Iis incorporated into the derivatized endotoxin which has a final minimum specific activity of ca. 2 - 5 mCi/mg based on a total recovery of column applied material. As it is not possible to accurately measure this small amount of LPS in the eluate by the TBA assay, the calculated specific activity is only a minimum estimate. We have, however, used a more direct approach to determine the specific activity by increasing the amount of LPS used in the labeling procedure. When a column is loaded with 170/zg pHPAA-LPS and the eluted fractions assayed by the TBA method, 8-10% of the starting material is recovered in one peak corresponding to Peak I. When the same amount of derivatized LPS is iodinated and fractionated, Peak I contains 8-10% of the starting material and Peak II, 26-30% of the starting material. Peak I therefore contains 21-33% of recovered radioactivity. (Data based on routine iodinations of this type carried out over a 2-year period.) 14F. C. Greenwood, W. M. Hunter, and J. S. Glover, Biochem. J. 89, 114 (1963).
270
TOXINS
[21]
These data suggest that (1) more iodide is incorporated into LPS when a larger amount is labeled and (2) 90% of the applied LPS sticks to the column nonspecifically, whether or not it is iodinated. On the assumption that unrecovered radioactivity is all in column-adherent LPS, we can estimate the amount of LPS recovered in Peak I and calculate the specific activity which is 2 - 5 mCi/mg. If the same fraction of LPS is recovered when 10 fig is iodinated, the actual specific activity could well be an order of magnitude higher.
Radioimmunoassay
Reagents 12 x 75 mm polystyrene tubes (Falcon No. 2052) Borate-buffered saline (BBS) with azide: prepared by adjusting pyrogen-free saline (0.9% NaC1, Travenol Laboratories) to 50 mM in borate and 0.02% NaNa at pH 8.0 ( " p H 8.0 BBS") and pH 9.5 ( " p H 9.5 BBS") Bovine serum albumin (BSA): 0.5% in pH 8.0 BBS (Sigma, RIA grade) IgG fraction of rabbit anti-LPS diluted in pH 9.5 BBS 37° shaker/water bath or a shaker (R-2 reciprocator, New Brunswick Scientific Co., set at 200 strokes/rain) in a 37° warm room
Procedure Coating of Tubes with Antibody. One-haft milliter of an appropriate dilution (see next section) of the IgG fraction of rabbit anti-LPS made up in pH 9.5 BBS is carefully pipetted into the bottom of each polystyrene tube. After 1 hr at room temperature the antibody solution is removed by aspiration and pooled for reuse. The tubes are incubated with 1 ml of 0.5% BSA for a further 30 rain at room temperature. After the BSA is discarded, the tubes are washed twice with 3 ml o f p H 8.0 BBS and drained inverted over absorbent tissue at 4°. Normally the tubes are used within a few hours, but they will maintain their binding capacity for 24-36 hr when stored at 4 °. The pooled antiserum has a shelf life of at least 1 month when kept at 4 °, but should be regularly monitored for 100% binding e ~ ciency if used repeatedly. Contamination with extraneous endotoxin is usually undetectable. Coating with 0.5% BSA does not normally improve the assay. Nonetheless, this procedure is recommended to preclude any unexpected nonspecific binding of I~SI-LPS to tube surfaces. Likewise, increasing the tube-coating time from 1 hr up to 4 hr does not appreciably increase the amount of IgG bound.
[21]
SOLID-PHASE RIA FOR BACTERIAL LPS
271
Determination of Antibody Titer. Anti-LPS IgG fraction is serially diluted in pH 9.5 BBS in 1 ml volumes in glass tubes. Polystyrene tubes are coated with 0.5 ml each dilution as described above. Coated tubes are subsequently incubated with ca. 15,000 cpm 125I-derivatized LPS in 600/zl pH 8.0 BBS and incubated with shaking for 4 hr at 37°. The solutions are removed by aspiration, the tubes are washed once with 3 ml of saline, and bound radioactivity determined in a gamma scintillation spectrometer. From a plot of percentage of added radioactivity bound against log10 concentration of anti-LPS IgG the antibody titer is estimated as the concentration of IgG which results in a 50% binding of tracer relative to the maximum observed binding. This IgG concentration is used in coating RIA tubes for detection of LPS. Detection ofLPS. LPS samples are serially diluted in pH 8.0 BBS in glass tubes. Four hundred microliters of each sample and 100/zl pH 8.0 BBS are incubated overnight at 4 ° in antibody-coated polystyrene tubes on a reciprocating shaker. One hundred microliters of 125I-derivatized LPS (ca. 15,000 cpm) are added, and the tubes are incubated for a further 2 hr at 37°. The reaction mixture is then removed by aspiration, the tubes are washed once with 3 ml of saline, and counted. Two controls are run concurrently: Bo (100% binding) contains 500/~1 of pH 8.0 BBS and 100/zl of ~25I-LPS; NSB (nonspecific binding) contains the same addition as B0 but uses tubes coated with 0.5% BSA only and no antibody. Samples and controls are run at least in duplicate, preferably triplicate. 10(3
60
20 •
~-I
0.0t
@
•
I
I-
.I.
.I
0.t
t.0
t0
100
~g/ml LPS FIG. 1. Detection of LPS in radioimmunoassay using IgG prepared on Affi-Gel Blue dextran.
272
TOXINS
[22]
Percentage bound is calculated from the relationship: Sample cpm - NSB cpm x 100% B0 cpm Standard curves are plotted as percentage tracer bound against log10 LPS concentration in/zg/ml (Fig. 1). LPS content of unknown is estimated from the dilution factor at 50% binding relative to that of the standard sample. In the case ofE. coli 055: B5 LPS, a sensitivity range of approximately 10-500 ng/ml was obtained) Nonspecific binding and 100% binding were about 5 and 60%, respectively, of the total counts added.
[22] R a d i o i m m u n o a s s a y s o f T h y r o x i n e (T4), 3 , 5 , 3 ' - T r i i o d o t h y r o n i n e (Ta) , 3 , 3 ' , 5 ' - T r i i o d o t h y r o n i n e ( R e v e r s e T 3 , rTa), a n d 3 , 3 ' - D i i o d o t h y r o n i n e (Tz) By W I L M A R M . W I E R S I N G A a n d I N D E R J . C H O P R A
Introduction Although there has been a decided improvement in our understanding of the field of endocrinology during the last decade because of the many rapid developments, the resulting increase in complexity of certain issues has led to confusion in some instances. For example, we now know that substances synthesized within an endocrine cell are not necessarily those conventionally viewed as the hormone, but merely are precursors (e.g., preproparathormone and proparathormone in case of parathormone). Along the same line, the substance secreted by an endocrine cell into the bloodstream is not necessarily the one that ultimately provokes the hormonal effect in the target tissues but only a prehormone (e.g., testosterone for dihydrotestosterone). The hormonal effects in the target cells may be modulated not just by circulating hormone levels but also by the receptor state (e.g., number and affinity of receptors for the hormone), and in some cases also by postreceptor alterations. Recent discoveries involving thyroxine (I"4) provide an excellent illustration of these complexities, First, T4 synthesized in the thyroid gland is secreted unchanged but is also converted intrathyroidally to a more active triiodothyronine (Ta). x Second, T4 released into circulation is monodeiodinated in the extrathyroidal tissues into either the more potent hormone i H. Ishii, K. Tanaka, K. Naito, M. Nishikawa, and M. Inada, Annu. Meet., 62nd, Endocr. Soc., Washington, D.C.p. 202, Abstr. 512 (1980).
METHODS IN ENZYMOLOGY, VOL. 84
Copyright© 1982by AcademicPress, Inc. All rightsof reproductionin any form reserved. ISBN 0-12-181984-1