Chapter 26
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Preparation and Characterization of Mouse Antibodies Against Hemoglobins Modified by Styrene Oxide Robert A. Haas, Doris Hollander, and Mitchell Rosner Air and Industrial Hygiene Laboratory, California Department of Health Services, Berkeley, CA 94704
Antibodies were raised which recognize styrene-7,8-oxide modified human hemoglobin. The covalent binding of styrene-7,8-oxide with human hemoglobin was measured at varying styrene oxide concentrations. The same reaction with Swiss-Webster mouse hemoglobin was carried out and resulted in a heterogeneous mixture of chemically-modified hemoglobins plus unreacted hemoglobin which was used without further purification to immunize BALB/c mice. Antibodies produced by this method cross-reacted with styrene-oxide modified human hemoglobin and demonstrated a preference for the chemically-modified hemoglobins. Each serum sample also cross-reacted to a slight but measurable extent with unmodified human hemoglobin but no cross-reactivity was observed using unmodified mouse hemoglobin. Antibody recognition of human hemoglobin required that the level of styrene oxide modification be greater than 0.03 styrene oxide residues per hemoglobin tetramer.
The analysis of chemically-altered hemoglobin (Hb) as a indicator of chemical exposure was pioneered by Ehrenberg and colleagues (1). Hemminki (2) demonstrated that the styrene metabolite, styrene-7,8-oxide (SO) (1,2-epoxyethylbenzene, CAS 96-09-3) became covalently bound to serum proteins and hemoglobin when incubated with human blood. This chemically-modified hemoglobin should be immunologically distinct from unmodified hemoglobin. Thus antibodies that recognize the modified Hb could be raised and utilized in an immunoassay to detect such molecules in blood samples from exposed individuals. This report describes the development of such antibodies in mice and their binding properties to styrene oxide-modified hemoglobins from mice and humans.
0097-6156/91/0451-0293$06.00/0 © 1991 American Chemical Society Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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M a t e r i a l s and M e t h o d s Reactions of styrene oxide ( S O ) with whole b l o o d in vitro were performed using freshly-drawn human b l o o d . T o determine S O incorporation into b l o o d , [ C]styrehe oxide (28 m C / m m o l , A m e r s h a m , A r l i n g t o n , I L ) i n 100% ethanol was added to 0.9 m l whole h u m a n b l o o d plus 0.1 m l sterile saline (0.9% N a C l ) to final concentrations of styrene oxide of 3.6 μ Μ , 36 μ Μ , 357 μ Μ , and 1.8 m M and incubated at 37° for 2 h. T h e reaction mixtures were centrifuged at 3000 χ g for 5 m i n and the pellets washed three times with 0.7 m l 0.9% saline. T h e red b l o o d cell pellets were lysed by addition of an equal volume of deionized water, the debris removed by centrifugation, and globin prepared by the acidic acetone method (3). S O incorporation was then determined by liquid scintillation counting of the redissolved globin pellet and expressed as n m o l S O per m l whole b l o o d assuming [ H b ] = 1 5 mg/ml (4). M o u s e and human hemoglobin were prepared by hypotonic lysis of red b l o o d cells, removal of stroma, and a m m o n i u m sulfate precipitation (50% saturation) o f contaminating proteins. T h e hemoglobin-containing supernatant was dialyzed against 0.01 M Bis-Tris ( p H 7.0) and used within one week. F o r preparation of immunogen and competitors, styrene oxide-modified hemoglobin was prepared by addition of an appropriate volume of a 5 M solution of [ C ] styrene oxide (50 D P M / n m o l ) to a solution o f 310 μ Μ human or mouse hemoglobin i n 10 m M Bis-Tris buffer ( p H 7.4) to yield the final desired concentration of styrene oxide. E t h a n o l (10% v:v) was added and the reaction mixture vortexed to aid i n solubilizing the styrene oxide. T h e reaction solutions were incubated i n the dark for 24 hours at ambient temperature. T h e styrene oxide/hemoglobin reaction mixtures were then chromatographed twice o n 150 μ η ι Sephadex G-25 sizeexclusion spun columns to remove the unreacted styrene oxide. T h e excluded volume containing the hemoglobin was dialyzed against three changes of water for 36 hours. A l i q u o t s of the hemoglobin were analyzed for radioactivity by liquid scintillation counting and for hemoglobin content by reaction with D r a b k i n ' s reagent to measure cyanmethemoglobin at 540 n m . T h e extent o f modification expressed as n m o l styrene oxide / n m o l hemoglobin tetramer was calculated. 14
w h o l e b l o o d
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T o raise antibodies against S O - H b , B A L B / c mice were i m m u n i z e d intradermally o n days 0, 7, and 21 with SO-modified mouse hemoglobin (1-2 S O residues/Hb tetramer) with 100 μ g conjugate i n 2 X R I B I ( R i b i Immuno c h e m i c a l R e s . Inc., H a m i l t o n , M T ) adjuvant. O n day 28, the mice were bled via the tail vein and the sera screened against SO-modified mouse and human hemoglobin and unmodified human hemoglobin. Ascites production was induced by intraperitoneal injection of A T C C sarcoma T G 1 8 0 (200 μΐ, « 1 0 cells). A competitive enzyme-linked immunosorbent assay ( E L I S A ) using S O modified H b as competitor was used to characterize the antibodies. Solutions for assay were prepared by addition of equal volumes o f a 1:100 dilution of the polyclonal ascites to an equal volume of the hemoglobin samples (at various dilutions) to yield the desired concentration of competitor. T h e control well 5
Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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contained only a 1:200 dilution of the ascites fluid. These solutions were prepared 12-16 h before the assay and stored at 4°. I m m u l o n 2 flat bottom 96 w e l l microtitration plates (Dynatech Laboratories, Chantilly, V A ) were coated with 0.5 μ g of the desired hemoglobin solution i n 100 μΐ phosphate buffered saline ( p H 7.2) at 4° for 16 h. T h e wells were aspirated, washed 3 times with 0.05% T w e e n 20 phosphate buffered saline solution ( P B S - T w e e n ) , and subsequently filled to capacity with a 1% bovine serum a l b u m i n i n P B S - T w e e n (v:v) and incubated for 0.5 h at r o o m temperature to block any uncoated sites in the microplate wells. After discarding the blocking solution, the anti body/competitor solutions were added to the appropriate wells. A f t e r 2 hours the wells were aspirated and washed 3 times with the P B S - T w e e n solution. T o each well was added 100 μΐ of a 1:1000 dilution of goat antimouse I g G alkaline phosphatase conjugate (Sigma C h e m i c a l C o . St. L o u i s , M O ) . F o l l o w i n g incubation for 2 h at 20°, the wells were washed 3 times with P B S - T w e e n before applying the alkaline phosphatase substrate (p-nitrophenyl phosphate, 4 mg/ml) solution i n 0.1% M g C l (w:v) diethanolamine buffer ( p H 9.8). T h e microplates were read at 405 n m either kinetically or after stopping the reactions at 1 hour with 50 μΐ of 0.1 M E D T A . 2
S e r u m titers were determined using plates coated as described above. S e r u m was obtained by tail bleeding. Results and Discussion T h e covalent binding of styrene oxide to human hemoglobin when S O is incubated with whole b l o o d or washed red b l o o d cells is shown i n Figure 1. O v e r the concentration range of 3 μ Μ - 1 . 8 m M S O , the amount of S O i n washed red b l o o d cells is approximately proportional to its initial concentration. In whole b l o o d , the linear range of S O incorporation into hemoglobin is [SO]=30 μ Μ - 1 . 8 m M . T h e ratio of S O / H b tetramer ranges from 0.0006-0.6 S O / H b tetramer. M u c h higher levels of modification may be achieved by lysing the erythrocytes and removing cell debris (5). W h e n this semi-purified human hemoglobin is reacted with S O at concentrations up to 50 m M , the styrene oxide reacts i n a linearly-related, dose-dependent manner similar to that seen for the whole b l o o d reaction. Concentrations of S O above 50 m M do not lead to additional modification but to extensive denaturation and precipitation of the protein. It appears that saturation of soluble H b occurs w h e n about five S O residues/Hb tetramer are achieved (unpublished observations). Semi-purified mouse hemoglobin was also reacted with S O to prepare immunogens. M o u s e hemoglobin (0.29 m M ) from Swiss-Webster mice reacts with S O (50 m M ) yielding 1.2 SO residues/Hb tetramer. This preparation was used to immunize B A L B / c mice. T h e serum titers of two such immune mice are shown i n Figure 2. T h r e e different immobilized antigens were used: (a) unmodified h u m a n H b ; (b) S O - m o d i f i e d h u m a n H b ; and (c) SO-modified murine H b (the immunogen). T h e antisera from both mice showed no cross-reactivity with unmodified human hemoglobin. T h e antisera from mouse #5 cross-reacts with modified human H b
Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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100 [styrene oxide]/^M
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Figure 1. Incorporation of [ C]-styrene oxide into whole b l o o d (open circles) and washed red b l o o d cells (filled circles) as a function of initial styrene oxide concentration. 100
1E5
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1000 1E4 Serum Dilution
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Figure 2. S e r u m titers of two individual B A L B / c mice (#5, Figure 2a and #1, F i g u r e 2b) i m m u n i z e d with styrene oxide-modified Swiss-Webster mouse hemoglobin. T h e antisera is titered against the immunogen, S O - m o d i f i e d S - W mouse H b , (filled squares), styrene oxide-modified h u m a n hemoglobin (filled triangles), and unmodified h u m a n hemoglobin (open squares).
Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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to about half the extent as the immunogen. M o u s e #1 antisera demonstrates approximately 100% cross-reactivity with SO-modified h u m a n hemoglobin. Injection o f a sarcoma cell line into the intraperitoneal cavity o f these i m m u n e mice provided a polyclonal ascites fluid. T h e use of antibodies i n the ascites fluid i n competitive E L I S A experiments using S O - m o d i f i e d h u m a n H b at different levels of modification is summarized i n Figure 3. In all cases the i m m o b i l i z e d antigen was 0.5 /zg of a high level (3.6 S O / H b ) modified h u m a n H b preparation. A s shown i n Figure 3, the degree of inhibition is related to the extent of modification. T h e H b with the lowest level of modification tested, 0.03 styrene oxide residues per tetramer, produced no inhibition even w h e n as m u c h as 500 μ g of the modified hemoglobin was incubated with antibody (data not shown). H i g h e r levels of competitor could not be used because inhibition of binding occurred even with unmodified hemoglobin as competitor at concentra tions greater than 100μg/well. This can be seen by the nearly 3 0 % inhibition that resulted using unmodified hemoglobin at 500 μg/well. Inhibition relative to unmodified H b is first seen at levels o f modification of 0.15 S O / H b . It may be that the lack of inhibition at lower levels o f modification is the result of too few binding sites at very low levels of modification, or there may be something qualitatively different about hemoglobin modified at different levels. T h e latter possibility is unlikely since peptide m a p p i n g of S O - m o d i f i e d hemoglobin from whole b l o o d reactions indicates that there is no single preferred site of reaction and that the pattern of modification o n several different peptides is qualitatively similar regardless of whether the hemoglobin is within the red b l o o d cell or not (manuscript i n preparation). T h e ascites fluid can be used for screening o f samples where the modification levels are high enough to permit detection, eg. i n animal studies employing relatively high S O doses. T h e r e is no significant cross-reaction of these antibodies with unmodified murine hemoglobin and thus these preparations can be used for in vivo mouse experiments, now i n progress. Summary and Conclusions M o u s e antibodies against SO-modified hemoglobin were raised using a heterogeneous immunogen consisting of chemically-modified mouse hemoglobin molecules i n a mixture that contained many different modified sites (5) as well as a large p r o p o r t i o n of unmodified molecules. It is shown that B A L B / c mice can mount an immune response to the SO-modified hemoglobin o f SwissWebster mice. A l t h o u g h there are amino acids differences between the /3-chains of these strains (6, 7), the chemical modification of hemoglobin provides the immunodominant epitopes as evidenced by a m u c h higher serum response to the modified hemoglobin. In addition, antibodies raised against styrene oxidemodified mouse hemoglobin also b i n d to similarly modified h u m a n hemoglobin. These mouse antibodies have a "threshold" of 0.15 S O residues per h u m a n H b tetramer below which no binding is observed.
Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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Competitor (ug) Figure 3. Competitive enzyme-linked immunosorbent assay. I m m o b i l i z e d antigen is styrene oxide-modified human hemoglobin (3.6 S O / H b tetramer), competitors are h u m a n hemoglobins modified at different levels: no modification (open circles), 0.15 S O / H b tetramer (filled diamonds), 0.4 S O / H b tetramer (filled triangles), and 3.6 S O / H b tetramer (filled squares).
Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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Acknowledgments We thank Jean Grassman for many helpful discussions and C. Peter Flessel for a critical review of the manuscript. Support by NIEHS grant ES04705 is greatly appreciated. Literature Cited 1. Tornqvist, M.; Mowrer, J.; Jensen, S.; Ehrenberg, L. Analytical Biochemistry 1986, 154, 255-266. 2. Hemminki, K. Arch. Toxicol. Suppl. 1986, 9, 287-290. 3. Ascoli, F.; Fanelli, M. R. R.; Antonini, E. In Meth. Enz.; Antonini, E., Rossi-Bernardi, L., Chiancone, E., Eds.; Academic: New York, 1981; Vol. 76, p 72. 4. Pereira, Μ. Α.; Chang, L. W. Chem.-Biol Interactions 1981, 33, 301-305. 5. Kaur, S.; Hollander, D.; Haas, R.; Burlingame, A. M. J. Biol. Chem. 1989, 264, 16981-12984. 6. Popp, R. A. Biochim. Biophys. Acta 1973, 303, 52-60. 7. ibid. 61-67. RECEIVED August 30, 1990
Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
