v>EPA
States
imental Protection
Environmental Research
Laboratory
Gulf Breeze FL 32561
EPA-600/3-78-093
October 1978
Research and Development
Determination of the
Site(s) of Action of
Selected Pesticides
by an Enzymatic-
Immunobiological
Approach
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1 Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7 Interagency Energy-Environment Research and Development
8. "Specia " Reports
9. Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-78-093
October 1978
DETERMINATION OF THE SITES (S) OF ACTION OF SELECTED
PESTICIDES BY AN ENZYMATIC-IMMUNOBIOLOGICAL APPROACH
by
Robert B. Koch
Mississippi State University
Department of Biochemistry
Mississippi State, MS 39762
Co-Investigators: W. E. McHenry, W. E. Choate, M. Barker,
B. R. Layton, R. Stinson, D. V. S. Subba Rao,
B. Click, T. N. Patil, and D. Desaiah
Grant No. R803458
Project Officer
Peter Schoor
Environmental Research Laboratory
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Environmental Research
Laboratory, Gulf Breeze, Florida, U.S. Environmental Protection
Agency, and approved for publication. Approval does not signify
that the contents necessarily reflect the views and policies of
the U.S. Environmental Protection Agency, nor does mention of
trade names or commercial products constitute endorsement or
recommendation for use.
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FOREWORD
The protection of our estuarine and coastal areas from
damage caused by toxic organic pollutants requires that regula-
tions restricting the introduction of these compounds into the
environment be formulated on a sound scientific basis. Accurate
information describing dose-response relationships for organisms
and ecosystems under varying conditions is required. The
Environmental Research Laboratory, Gulf Breeze, contributes to
this information through research programs aimed at determining:
°the effects of toxic organic pollutants on individual
species and communities or organisms;
°the effects of toxic organics on ecosystem processes and
components;
°the significance of chemical carcinogens in the estuarine
and marine environments.
In understanding of toxicological processes it is of utmost
importance that the mechanisms responsible be known. This
report deals with one of these processes: the response of an
organism to a compound recognized as foreign by the immune sys-
tem. While this mechanism is probably of little consequence in
acute poisoning, compounds such as DDE with long residence times
in the blood stream may ultimately be deactivated through the
possible action of antibodies formed as a response to the toxi-
cant. As work of this type continues, there should be an answer as
to whether or not the already established detoxification/excre-
tion processes or elimination of toxic compounds from an organ-
ism can be supplemented by the immune^system.
Thomas W. Duke
Director
Environmental Research Laboratory
111
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ABSTRACT
This research program was initiated with the overall objec-
tive of producing an antibody to an organochlorine pesticide
which would be used in studies on its mechanism of inhibition of
the ATPase system from various sources. A single pesticide
(Kelevan) was chosen for use as a hapten for covalent conjugation
to various protein antigens. The approach to this study included
organic synthesis and instrumental analysis, antibody production
and detection by immunological procedures, and biochemical inves-
tigations to determine the effects of antibody on the inhibition
of the ATPase system by Kepone, a derivative of the hapten,
Kelevan.
Kelevan, the condensation product of ethyl levulinate and
Kepone, was successfully conjugated to bovine serum albumin
(BSA), fibrinogen (BF), and gamma globulin (BGG). Rabbits and
chickens preimmunized with BSA and then immunized with BSA-
Kelevan produced antibodies to both the hapten, Kelevan, and the
carrier protein BSA. Antiserum to Kelevan protected ATPase
activity against Kepone and its derivatives. The titer of anti-
body to Kelevan was critical since antiserum with only trace
amounts of Kelevan antibody failed to protect the ATPase activity
against Kepone inhibition.
Antibody was concentrated by Na2S04 fractional precipitation
of the antiserum and obtained in pure form by affinity chroma-
tography using BGG-Kel covalently linked to Sepharose 4B. Pure
antibody was obtained from untreated blood serum or plasma with
no prior pretreatment or fractionation using the BGG-Kel affinity
column. Complete protection of mitochondrial Mg2+ATPase activity
from in vitro inhibition of Kepone was obtained using a 1.2 mg
quantity of Na2S04 fractionated antibody and only 120 yg of pure
antibody. Reversal of ATPase inhibition was readily obtained by
addition of antibody prior to addition of substrate to the
reaction mixture. In a brief study conducted after the comple-
tion date of this grant, it was observed that the antibody to
Kelevan was not specific for Kepone but also prevented inhibition
by other chlorinated hydrocarbon pesticides. The possible
meaning of this observation is briefly discussed.
This report was submitted in fulfillment of Grant No.
R803458 by Mississippi Agricultural and Forestry Experiment
Station, Mississippi State University under the partial
IV
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sponsorship of the U. S. Environmental Protection Agency. This
report covers the period from February 15, 1975 to February 14,
1978; work was completed as of April 28, 1978.
v
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CONTENTS
Foreword ill
Abstract iv
List of Abbreviations vii
Introduction 1
Conclusions and Recommendations 3
Chemistry Studies 5
Materials and Methods 5
Results and Discussion 7
Immunology Studies 11
Materials and Methods 11
Results and Discussion 13
Biochemistry Studies 1.6
Materials and Methods 16
Results and Discussion 17
References 27
VI
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Kepone (Kep)
Kelevan (Kel)
Reduced Kepone
(DCPD)
BSA
BGG
BF
KS
LIST OF ABBREVIATIONS
— [1, 2 , 3, 4, 6, 7, 8, 9,10, 10-decachloro-pentacyclo
( 5 . 3 . 0 . 0 *'6 . 0 3'9 . 0 * * ) -decan-5 . 5 dihydroxide ]
--[ethy] 6- (-5-hydroxyl-l, 2 , 3, 4 , 6, 7, 8 , 9 , 10 , 10-
decachloro-pentacyclo- {5.3.0.026.039.048} decyl)
levulinate] .
— [1 , 2 , 3 , 4 , 6 , 7 , 8 , 9 , 10, 10-decachloro-pentacyclo
( 5 . 3 . 0 . 0 *'6 . 0 3'9 . 0 M ) -decan-5-ol ] .
--Bovine serum albumin
— Bovine gamma globulin
— bovine fibrinogen
--Kelevan N-hydroxysuccinimide
VII
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INTRODUCTION
During studies on the ATPase systems, Koch (1964, 1969/70)
observed and reported sensitivity to organochlorine pesticides
at micromolar concentrations. He suggested that inhibition of
the ATPase enzymes by organochlorine pesticides could be associ-
ated with the mechanism of toxic action of these compounds. Work
has continued in Koch's laboratories in conjunction with a number
of colleagues but especially in the laboratory of L. K. Cutkomp
and with the excellent collaboration of D. Desaiah. Of the over
thirty papers published by these authors on this topic of ATPase
sensitivity of organochlorine pesticides, the following selected
references strongly indicate the relationship between ATPase
inhibition and mechanism of toxic action (Desaiah and Koch, 1975;
Desaiah, e_t al. , 1975; Cheng and Cutkomp, manuscript submitted;
Koch et al., 1977; other references by these authors are given
in these papers).
Also in vivo studies have been reported which indicate that
mitochondrial Mg2+ATPase inhibition occurred in fish brain and
rat liver. In an iri vivo study on DDT inhibition of ATPase
activity in fish Pimephales promelas, specific inhibition of
mitochondrial (Mito) Mg^+ATPase activity in brain was observed
(Desaiah et al., 1975) . Although a number of exposed fish died
prior to completion of the study, the maximum inhibition observed
for this enzyme activity in the surviving fish after 266 days
exposure was only about 60%. Therefore, the 50-60% inhibition of
brain oligomycin sensitive (Mito) Mg2+ATPase activity was con-
sidered sufficient to cause death, or a partial reactivation of
the enzyme activity occurred during homogenization and fractiona-
tion procedures. A similar phenomenon was also observed in liver
preparation of rats fed with Kepone for 16 days (Desaiah et al.,
1977).
To obtain more definitive information on their mode of
action, it was proposed to produce an antibody to an organo-
chlorine pesticide. The antibody with high affinity for its
specific antigen would be used to gain insight into the nature
of the interaction of the enzyme complex with the pesticide.
This final report presents the results of a three-year study on
this project under partial support by EPA Grant R803458 (from
February 15, 1975 to February 14, 1978).
The,use of pesticides as haptens for antibody production
has been reported by Anderson et al., (1964), Langone and Van
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Vunakis (1975), Cento et al. (1970), and Haas and Guardia (1968).
The high specificity of antibodies for haptens (in this case
pesticides conjugated to antigenic protein) have caused interest
in their use for the development of highly sensitive radioimmuno-
assay procedures for pesticides (Langone and Von Vunakis, 1975).
In studies on the mode of action of organochlorine pesti-
cides, it was shown that Kepone and its monohydroxy-reduced
derivative strongly inhibited ATPase system in fish brain
preparations (Desaiah and Koch, 1975a). Further, because of the
availability of a highly purified derivative of Kepone, Kelevan
(an analytical standard grade), it was proposed to use Kelevan
as the hapten to be covalently linked to the antigen BSA.
This final report is divided into three parts according to
the discipline required to conduct the three different aspects
of the research program: I Chemistry Studies; II Immumology
Studies; and III Biochemistry Studies.
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CONCLUSIONS AND RECOMMENDATIONS
An antibody to the pesticide Kepone was produced in rabbits
and chickens. The antigen-hapten complex (BSA-Kelevan) used
for stimulation of antibody production was synthesized by
reacting BSA with the succinimide derivative of Kelevan. In
addition, Kelevan derivatives were prepared using BGG and BF
following the same procedures. The latter two derivatives were
used for the detection of antibody in blood serum by the
micro-ouchterlony immunodiffusion test.
An affinity column was prepared by covalently linking BGG-
Kel to a cyanogen bromide activated Sephrose 4B support matrix.
Purified antibody was prepared by passing blood serum directly
through the column. The affinity bound antibody was removed
by 2M KCl buffer solution. Antibody obtained from the affinity
column was shown to be highly active in preventing or reversing
inhibition by Kepone. An unexpected result was observed in that
the antibody also protected against other organochlorine
pesticides (DDT and aldirin) . However, the antibody did not
reverse the inhibition by the pesticide Plictran (a non-
chlorinated compound).
It is concluded from the latter result that the antibody
exerts its action on organochlorine type structures rather than
the particular compound which was used as the hapten. This
result may indicate that inhibition of the ATPase system by
organochlorine pesticides occurs through a similar mechanism,
such as disruption of secondary bonding forces of the lipo-
protein complex of the enzyme system.
It is recommended that further studies be conducted using
the antibody Kelevan to determine its effect on inhibition by
other organochlorine pesticides and using different tissue
sources for the ATPase system. It is also recommended that
at least one additional antibody to an organochlorine pesticide
(such as DDT or Kelthane) be produced to compare the effects
of such an antibody on the inhibition by Kepone and other
organochlorine pesticides. If the two antibodies have similar
effects, this would indicate that the antibody's specific
binding site is not just directed toward a particular compound
but also directed towards a type of structure in that compound.
Antibody cross-reactivity by similar structures is not sur-
prising, but the molecular structures of Kepone and DDT would
appear to be quite different. However, molecular modeling of the
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two compounds to observe for 3-dimensional configurations could
prove to be important. Such information, if this could be
obtained from the above studies, might yield information
concerning why compounds like DDT and Kepone exert different
intensities of inhibition of the ATPase system; namely, DDT
is more effective on mitochondrial Mg^+ATPase than Kepone.
It is also recommended that studies be conducted on
experimental animals exposed under in vivo conditions to Kepone
(or other selected pesticides) to determine whether there is a
"target" tissue for the pesticide. These studies would include
removal of tissue after in vivo exposure to sublethal concen-
trations of Kepone for varying periods of time. The tissues
would be homogenized, fractionated and ATPase activity deter-
mined, followed by measurement of reversal of ATPase activity by
exposure to the pure antibody to Kepone. The tissue showing the
greatest reversal of inhibition would be the most sensitive site
of action of the pesticide. This information would be useful
in designing stress or behavioral tests which would indicate
reduced physiological capacity due to pesticide toxicity.
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CHEMISTRY STUDIES
Materials and Methods
Conjugation of Kelevan to Bovine Serum Albumin (BSA)
The procedure chosen to functionalize the hapten and to
conjugate it to BSA is as follows:
0
dicyclohexyl
carbodiimide
cone.
HC1
J-COOH
II
BSA
buffered
0
III
-NH BSA
IV
Kelevan (I) [ethyl 6-(5-hydroxy-l,2,3,4,6,1,8,9,10,10-deca-
chloropen tacyclo{5.3.0.()2,6o. 3,9().4,8}decyl) levulinate] was
first hydrolyzed to the free acid (II) with concentrated hydro-
chloric acid. The free acid was then treated with N-hydroxy-
succinimide to produce the ester (III). This type of ester has
previously been shown to rapidly react with the amine groups of
araino acids to yield amides (Anderson e_t al., 1964) and with
free amine groups of protein to yield hapten-protein conjugates
(Langone and Van Vunakis, 1975). Therefore, treatment of III
with BSA was expected to produce the desired conjugate (IV) by
forming amide bonds with the free groups of the lysine units of
BSA. Kelevan was supplied by the Allied Chemical Corporation.
All other reagents and solvents were purchased from commercial
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sources. Melting points were determined on a Mel-Temp apparatus
and are uncorrected while infrared spectra were determined on a
Perkin-Elmer Infracord in KBr wafers.
Functionalized Hapten
To a solution of Kelevan (1) (52.9 g; 0.1 mole) in 50 ml of
ether was added 150 ml of concentrated hydrochloric acid. The
resulting solution was heated to reflux for 6 hr. The solution
was concentrated until the free acid separated as an oil. Puri-
fication of the crude product was achieved by dissolving the oil
in ether, drying the solution with molecular sieve (8-12 mesh),
and removing the solvent. By this procedure 39 g (78%) of
purified acid was obtained [infrared absorption for C-OH at 3400
cm"1 was strong and broad and for the carboxyl group at 1700
cm"1 was strong].
To a cooled solution containing a mixture of the free acid
of Kelevan (39 g; 0.78 mole) and of N-hydroxysuccinimide (8.97
g; 0.78 mole) in 250 ml of dioxane was added 15.9 g (0.78 mole)
of n, N-dicyclohexylcarbodimide. The reaction mixture was
allowed to stand overnight in the refrigerator. The precipitated
dicyclohexylurea was collected, washed with dioxane, and the
combined filtrate and washings were concentrated until all of
the solvent was removed. Infrared analysis of the residue showed
it to be a mixture of desired derivatives and dicyclohexylurea.
Repeated crystallizations of the mixture from methylene chloride
and 1-propanol gave the desired derivative as colorless crystals,
m.p. 2450, infrared absorptions for C=0 at 1700, 1740, 1790, and
1830 cm"1 and for C-OH at 3340 cm"1.
Conjugation of Functionalized Hapten for Immunization
To a solution of 100 mg of BSA in 100 ml of water (buffered
to pH 7.1) was added 114 mg of functionalized hapten dissolved
in 5 ml of tetrahydrofuran. (Previous control experiments
indicated this amount of tetrahydrofuran [THF] would not denature
the protein). The mixture was then stirred and cooled for 30 hr.
The Kelevan-protein derivative containing mixture was concen-
trated by freeze-drying. The residue was redissolved in water
or 0.1M NaCl, passed through a Sephadex G-25 column and the 280
nm peak from the column was collected and freeze dried. The 280
nm absorbance indicated separation of the total protein from the
other components in the conjugation reaction. The freeze dried
preparations were analyzed for Kelevan and used for immunology
studies.
Analysis of the Kelevan-BSA Conjugate
A method for analysis of Kelevan in protein samples was
developed. This method had high sensitivity (microgram levels)
and was capable of detecting Kelevan bound to protein. The
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analysis was accomplished by oxidizing both the protein and
Kelevan (which oxidizes to Kepone) and analyzing for Kepone. In
an oxygen atmosphere at 400°C, Kelevan is oxidized to Kepone;
whereas, the protein is oxidized to various gaseous products. A
sample (1-3 ing) was placed in a 25 ml ampule. The ampule was
evacuated to 0.5 mm of mercury pressure, filled with oxygen
(160 mm of mercury pressure), and then sealed. The sealed ampule
was placed in an oven (400°C) for five minutes and then removed
and allowed to cool. After the cooled ampule was opened, the
Kepone was extracted by washing the ampule several times with
methanol and petroleum ether. The extracts were combined,
diluted to a standard volume, and analyzed for Kepone on a gas
chromatograph equipped with an electron capture detector.
The concentration of Kelevan initially present in the
sample was calculated from the amount of Kepone found after
oxidation. The efficiency for oxidizing Kelevan to Kepone
(approximately 30%) was determined for each set of samples by
including one sample which contained the protein (1-2 mg) and
Kelevan (1-2 mg).
Preparation of a Kelevan-Bovine Gamma Globulin Conjugate for
Specificity Studies
In order to determine if the antibody for Kelevan-BSA was
specific for Kelevan, Kelevan-BGG and Kelevan-BF were prepared.
The procedure employed for conjugation was identical with that
used for preparing the Kelevan-BSA conjugate.
Results and Discussion
Pure Kelevan was available as analytical standard. It
was selected as a Kepone derivative for conjugation to protein
because the levulinate group of Kelevan could act as a means of
extending the hapten groups from the large protein molecule.
The use of such a "bridge" is common to enhance the antibody
production to the hapten (in this study the hapten is related
to Kepone and to the 5-monohydroxy reduced derivative of Kepone).
Conjugation of Kelevan to BSA by the procedure outlined in
the Materials and Methods, i.e., adding functionalized hapten in
a THF solution and purification of the conjugate by freeze-drying
and Sephadex G-25 chromatographic separation, yielded the sample
for immunization studies. Figure 1 and Table 1 give an example
of the 280 nm absorbance curve and Kelevan content of fractions
from G-25 gel filtration.
Analysis of several Kelevan-BSA conjugates as described in
the Materials section gave values of Kelevan in the samples of
2.5 ± 0.5%. These results are the average of three runs. In
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earlier preparations, other sets of duplicate runs gave values
as high as 15% Kelevan (probably due to contamination by uncom-
bined Kelevan) in the conjugate and as low as 0%. To trace the
source of the variation in the results, several standard runs
were made in which various parameters were changed. The
variables in the results were found to be due to any remaining
imidazole (used as a buffer in the synthetic step) in the
samples which had been purified by Sephadex G-25 column chroma-
tography and to the time allowed for the conjugation reaction.
If known amounts of Kelevan and imidazole are placed in a sealed
ampule and treated by the procedure described for analysis, no
Kelevan is found by g.c. analysis. Apparently, imidazole
totally dechlorinates the Kelevan and, therefore, the electron-
capture detector on the g.c sees no product. This could mean
that the 2.5 ± 0.5% values for Kelevan in the conjugate were
minimum values. Because of these results, the buffer used for
the conjugation reaction was changed to 0.1M sodium phosphate pH
7.1. Yield of BSA-Kel was slightly reduced but problems with
Kelevan analysis were eliminated.
Results of analysis of preparations made during 1977 using
phosphate buffer are given in Table 1. Only preparations which
showed the presence of approximately 2.5% Kelevan conjugated to
BSA were used for injection in animals for antibody production.
W. E. McHenry
W. E. Choate
M. Barker
Chemistry Department
B. R. Layton
Mississippi State Chemical Lab,
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TABLE 1. KELEVAN CONCENTRATION (%) IN VARIOUS
PROTEIN-KELEVAN CONJUGATES
BSA-Kel
Tube #
3-5
6
7
8-12
13-15
19-27
Kelevan
2.6%
3.4%
0.5%
0.02%
0.1%
55.0%*
BGG-Kel** BF-Kel**
Tube # Kelevan Tube # Kelevan
2-3 0.1% 2-3 0.1%
4-6 0.4% 4-6 0.5%
7-9 0.7% 7-10 0.25%
*Nonconjugated Kelevan.
**Fractionation not considered satisfactory because considerable
material was lost due to insolubility of protein due to con-
centration of solutions from chromatographic separation.
Therefore, BGG-Kel and BF-Kel were purified by extensive
dialysis against distilled water. The resulting products
contained 1.1% Kel (BGG-Kel) and 2.4% Kel (BF-Kel) some of
which may have been non-covalently bound.
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Chromatographic Separation of BSA-Kel
T
>£.
i
|4
i-o &
15 20
Tube*
Figure 1. Separation of components in BSA-Kelevan conjugation reaction mixture.
(THF removed on roto-evaporator; mixture centrifuged 18,000 x g for 10 min.;
supernatant concentrated by freeze drying; solubles from thawed samples separated
by using Sephadex G-25 Chromatographic column (l.S x 30 cm) using distilled water
or 0.1M NaCl as eluent; 5 to 7 ml fractions collected) . Tube 3-6 had a distinct
yellowish color not present in the other tubes (see Table 1). Tubes 19-23
combined and the high concentration of Kelevan to BSA (55%) indicated the
presence of unbound Kelevan.
10
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IMMUNOLOGY STUDIES
Materials and Methods
Antiserum Preparation
Three rabbits and fifteen New Hampshire chickens were
primed with soluble bovine serum albumin (BSA) at week zero.
One week later, the rabbits were supplementally immunized in the
footpads with a complete Freund's adjuvant (CFA) emulsion
containing 5 mg/ml BSA-Kel conjugate. All rabbits were injected
subsequently in weeks 6, 9, and 16.
Blood was collected from the marginal ear vein from both
control and experimental rabbits in weeks 7, 11, and 18. The
blood was allowed to clot at room temperature for 1 hour, the
clot ringed, and the serum allowed to separate overnight at 4°C.
The serum was centrifuged (10 minutes at 1200 rev/min) and
stored at 4°C.
A slightly modified procedure was employed with the chicken.
One week after the primary injection, the chickens were supple-
mentally immunized intramuscularly with CFA emulsion containing
5 mg/ml BSA-Kel conjugate. All chickens were injected subse-
quently in weeks 6, 9, and 16.
Blood was collected by cardiac puncture from both control
and experimental birds in weeks 7, 11, and 18. The blood was
allowed to clot for 1 hour, the clot ringed, and the serum
allowed to separate overnight at room temperature. The serum
was centrifuged (10 minutes at 1200 rev/min) and stored at 4°C.
Both the rabbits and chickens were immunized using an
adjuvant modified antigen. Adjuvants can be described as
substances that when mixed with an antigen prior to injection,
enhance antibody production. They are particularly useful if
the antigen is of low immunogenicity. Freund's complete
adjuvant is a water-in-oil emulsion, composed of antigen in
saline and a mixture of an emulsifier with Mycobacteria
(Campbell et al., 1970).
In the last year of the grant, we utilized the chicken more
extensively than the rabbit because chickens are better producers
of precipitating antibody than are rabbits (Wolfe e_t al. , 1942,
1957, 1960). For example, it has been shown that rabbits produce
11
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only raicrogram amount of precipitating antibody protein per
milliliter of serum against human hemoglobin after extensive
injections, whereas chickens produce as much as 3 mg of
precipitating antibody protein per milliliter of serum after only
4 intraperitoneal injections of the same antigen (Goodman and
Campbell, 1953).
Ouchterlony Double-Diffusion (Theory)
In double diffusion, antigen and antibody migrate toward
each other through gel which originally did not contain either
of these reagents. As the reagents come in contact with each
other, they combine to form a precipitate that is trapped in the
gel matrix and immobilized. This test offers the unique
advantage of not only enumerating the minimum number of antigen-
antibody systems reacting in a given mixture, but also indicating
the relationship among various antigens. This type of test
depends on the specific precipitation of antigen-antibody
complexes at the proper ratios and on the solubility of the
precipitate in excess of antigen or antibody. Only those
antigens that migrate through the gel and for which antibodies
are present in sufficient concentration in the antiserum can
form lines of precipitation.
Four basic patterns can be obtained with an antiserum and
preparations of single antigens.
I. Pattern of identity - both antigens have a set of
identical determinant groups with respect to the antiserum
employed.
II. Pattern of nonidentity - antiserum does not contain
antibodies to any determinant groups common to both
antigens, and thus the two lines formed are completely
independent of each other and cross without interaction.
III. Partial identity - one antigen (Basic Patterns III, below)
reacts more fully with the antibodies employed, while the
other antigen reacts with fewer antibodies, allowing
formation of a "spur."
IV. Two antigens have at least one type of determinant group
in common and, in addition, each has antigenic groups
not shared by the other. Thus the reaction obtained is
that of partial coalescence on both sides and formation
of double spurs (Williams and Chase, 1971).
Depending on concentration of antibodies and antigens
involved, many modifications of these four basic patterns can
be obtained.
12
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Basic patterns
Basic precipitate patterns obtained in double gel
diffusion. I. Complete coalescence. Reaction of
identity. II. Absence of coalescence. No cross-
reaction. III. Partial coalescence of one antigen.
Cross-reaction. IV. Partial coalescence of two
antigens.
Antibody Protection
A micro-adaption of Ouchterlony's technique (Auernheimer
and Atchley, 1962; Brewer, Click, and Vinson, 1972; and Click,
1976) was utilized to determine the presence of specific
antibody to Kelevan. Antigens used in the study were BSA, BSA-
Kelevan, bovine gamma globulin (BGG), BGG-Kelevan, Kelevan,
fibrinogen-Kelevan, and fibrinogen (FB) .
The central well was charged with antisera and the
peripheral wells with antigens and allowed to stand for 24-48 hr
at 25OC, for maximum precipitin line development. The templates
were then removed and the slides placed in physiological saline
(0.9%) for 48 hr. The slides were then rinsed in distilled
water for 10-15 minutes and placed in 20% acetic acid for 1 hr.
The slides were stained for 90 sec in 2 ml of 0.1% Amido
Schwartz 10B. They were rinsed in distilled water and allowed to
air dry.
Results and Discussion
Antibody Production to Kelevan
Preimmunization of rabbits with a carrier protein prepares
the animal for markedly enhanced antibody production to a second
molecule conjugated to that carrier (Katz et al., 1970 and Paul
et al., 1970). Their studies indicate that the carrier
13
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specificity of hapten-specific anamnestic antibody responses is
largely due to the interaction of two independent cell-associated
recognition units, one specialized for carrier and the other
specific for hapten determinants.
All experimental rabbits and chickens were preimmunized
with the carrier protein, BSA, in saline and subsequently
received primary immunization of 1.0 mg/ml of BSA-Kelevan
emulsified in CFA. Subsequent inoculations were made according
to the protocol outlined in the Methods section.
Separate antibodies are produced to BSA and to Kelevan in
a hapten-carrier conjugate. An affinity column to Kelevan
conjugated to a protein other than BSA will selectively remove
antibody to Kelevan. When this isolated antibody was tested by
the micro-Ouchterlony procedure, precipitin bands appeared where
Kelevan was as an antigenic determinant regardless of the carrier
molecule (i.e., BSA, BGG, or FB) [Fig. 2, Card D]. This was
true whether the antibody was produced in a rabbit or chicken.
On the other hand, if serum from a BSA-injected animal was
reacted against the same antigens, precipitin bands developed
against BSA and not against Kelevan (see Fig. 2, B).
R. Stinson
D. V. S. Subba Rao
B. Click
Poultry Science Department
14
-------
Month Temp. Hr
R 24
R
24
1-CT
24
00
of/6
o o
B
j'
o o
8S/1 O O OFJB-K/
Figure 2. Micro-Ochterlony precipitin reactions of antibodies
from blood serum from BSA and BSA-Kel immunized rabbits and
chickens. A. Blank slide indicating the positions of various
solutions antigen components. Antibody preparation was placed
in the center well. B. Antibody from chicken (serum) immunized
only with BSA. C. Antibody from rabbit (serum) immunized with
BSA-Kel. D. Antibody from chicken (serum) immunized with BSA-
Kel.
15
-------
BIOCHEMISTRY STUDIES
Materials and Methods
Kepone and its reduction product, DCPD, were chosen as
representative pesticides because of their broad inhibitory
action (in vitro) on the ATPase system (Desaiah and Koch, 1975a)
and the availability of Kelevan (as mentioned in an earlier
section). The covalently linked BSA-Kel was used as the antigen
for antibody production (see previous section). Kepone and
Kelevan were obtained from Allied Chemical Company and were
recrystallized. DCPD was prepared at the Mississippi State
Chemical Laboratories. Purity of all samples was 98+%.
Serum from control and immunized rabbits was used for
preparation of immunoglobulin (Ig) by the procedure of Kulkarni
and Click (1974). The ;second 14% (w/v) Na2S04 Ig sediment
(9,000 x g for 10 min) was suspended at similar protein levels
(1.2 mg/ml) in 0.1M imidazole buffer, pH 7.5.
Sepharose 4B was used as support material for preparation
of a BGG-Kel affinity column. Sepharose 4B was reacted with
CNBr in alkaline solution according to the procedure of Axen as
described by Cuatrecasas (1969). The Sepharose 4B-CN prepara-
tion was slowly stirred overnight at 4° after addition of BGG-
Kel. BGG was thus covalently linked to Sepharose 4B. After
washing, the complex was used to prepare an affinity column
(2X30 cm) for specific binding antibodies to the hapten (Kel)
produced in response to the antigen BSA-Kel. Blood serum or
plasma from immunized rabbits or chickens (4 to 6 ml) were
allowed to penetrate into the top of the affinity column
(previously washed with 0:ImM Tris-HCl pH 7.0) and the unbound
constituents were eluted with lOmM Tris-HCl pH 7.0. When the
effluent from the column showed zero absorbance at 280 nm, the
eluting solvent was changed to 2 M KC1 in 10 mM Tris-HCl, pH
7.0. The effluent was collected in 5-7 ml fractions and the
tubes containing 280 nm absorbing material were combined; and
then concentrated and wasried with 10 mM Tris-HCl using an XM-50
Diaflo filter in an Amicon Stirred Ultrafiltration Cell using
N2 pressure at about 10 psi.
Sources of tissue for preparation of 13,000 x g sediment
(mitochondrial and nerve ending particle) of subcellular particle
fractions from homogenates (in 0.32M sucrose, 10 mM Imidazole
buffer pH 7.5, 1 mM EDTA), were fire ant head, dog brain cortex,
housefly head and thorax, and mice and rat hearts. Homogenization
16
-------
and fraotionation were as reported by Koch (1969a). Protein
content of each fraction was determined by the method of Lowry
et al. (1951) using BSA as the standard. Samples were then
diluted to contain 15 to 25 yg protein per 25 to 50 yl homogenate
fraction.
ATPase activity was measured by the continuous method of
Pullman et al. (1960) and Fritz and Hamrich (1966) as reported
by Koch (1971/72). A 1-ml reaction mixture contained components
at the following concentrations: 4.5 mM ATP, 5 mM Mg2 + , 100 mM
Na+, 20 mM K+, 135 mM imidazole buffer (pH 7.5), 0.19 mM NADH,
0.5 mM PEP, 0.02% BSA, approx.9 units pyruvate kinase and 12
units lactic dehydrogenase, and 50 yl homogenate fraction (15-25
yg protein). Absorbance changes were measured at 340 nm for
10 min using a Gilford recording spectrophotometer with tempera-
ture controlled at 37°C in reaction mixture. Total ATPase
activity was determined in the presence of Na+, K+ and Mg2+.
Mg2+ ATPase activity was measured using the same mixture plus
ouabain at a concentration of 1.0 mM. Na+-K+ATPase activity is
total activity minus Mg2+ATPase activity. Oligomycin (2 yg/ml in
the reaction mixture) was used to delineate oligomycin-sensitive
(mitochondrial) and insensitive Mg2+ATPases. The data obtained
were based on the duplicate determinations. The variation in
the replicate values was less than 5% of the average. Absorbance
at 340 nm per 10 min per 10 yg protein at 37°C used for measure-
ment of ATPase activities. Specific activity = ymoles Pi mg~l
protein hr~l. Oligomycin used to determine mitochondrial Mg2+
ATPase (oligomycin sensitive) and Oligomycin insensitive
activities.
Pesticides used in inhibitor studies were added as ethanol
(95%) solution. The solutions were prepared so that a 1 yl
addition gave the desired pesticide concentration in the reaction
mixture. The ethanol solution was injected into a rapidly
stirring reaction mixture (using a Jr. Vortex mixer) with a
Hamilton 1 yl microsyringe. Protein-Kel derivatives and
antibody preparations were added to the reaction mixture using
10-100 yl microsyringes or micropipetters.
Results and Discussion
Initially the compounds related to Kepone, which were to be
used in the covalent binding to BSA, were evaluated as inhibitors
of the ATPase activities from a fire ant head homogenate frac-
tion. Table 2 shows that Kelevan (the levulinate derivative of
Kepone) was much less effective than Kepone or DCPD. However,
Kelevan caused over 50% inhibition of mitochondrial Mg2+ATPase
activity at 6 x 10~5M. A BSA-Kel preparation was also tested
and found to cause inhibition of fire ant head ATPase activities
(molar concentration of Kel in the BSA-Kel was estimated to be
about 20 yM).
17
-------
TABLE 2. COMPARISON OF SEVERAL COMPOUNDS RELATED TO KEPONE
FOR THEIR INHIBITION OF ATPASE ACTIVITIES FROM A FIRE ANT
HEAD HOMOGENATE FRACTION
Addition __^_^ Per Cent Inhibition
Na+-K+ ATPaseM^+ ATPase
Oligomycin
Sensitive Insensitive
Kepone (15 yM)
Kelevan (60 yM)
KS* (20 yM)
DCPD (7.5 yM)
73
31
75
36
100
52
82
92
25
42
21
20
*KS - Kelevan N-hydroxysuccinimide.
Table 3 shows the results from the first test of a crude
antiserum (used without fractionation or purification) compared
to control antiserum. The antiserum had been shown to contain
antibodies to Kelevan by the Micro-Ouchterlony immunodiffusion
test (see Immunology Studies). It was clear from this test that
the antiserum could prevent the inhibition of ATPase activities
by DCPD (Table 3).
A large sample of antiserum was obtained and fractionated
with sodium sulfate (see Materials and Methods). The immuno-
globulin (Ig IV) fraction was tested for protection (Table 4,
Ig added with DCPD before enzyme) and reversal (Table 5, Ig
added 5 min after combining enzyme and Kepone) of ATPase activi-
ties inhibited by organochlorine pesticides.
18
-------
TABLE 3. EFFECT OF RABBIT ANTISERUM TO BSA-KEL ON THE
INHIBITION OF FIRE ANT HEAD HOMOGENATE ATPASE
ACTIVITIES BY DCPD
Addition
Per
Na+-K+ ATPase
Cent
Inhibition
Mg^+ ATPase
Oligomycin
Sensitive Insensitive
DCPD (7.5 yM) *
Antiserum (50 yl)*
Antiserum (50 yl)**
+DCPD (7.5 yM)
Control Serum* (50
Control Serum*** (50
+DCPD (7.5 yM)
36
26
0
yl) 15
yl) 16
92
27
0
0
54
20
+ 45
0
+ 33
26
*Compared to control
**Compared to antiserum only activities
***Compared to control serum only activities
TABLE 4. EFFECT OF RABBIT ANTISERUM* TO BSA-KEL ON
DCPD INHIBITION OF FAH (MS)
FAH (MS )
Additions
ATP +
ATP +
ATP +
ATP +
Enz
DCPD
Ig IV
DCPD-*
Na^
(7.5ym)-*Enz
(1.2mg)-*Enz
Ig IV+Enz
L-K+
.100
.003
.009
.084
ATPase Activities
Mito
.041
.003
.030
.012
Oligo
.040
.046
.032
.028
*Antiserum was fractionated and the immunoglobulin
fraction (IglV) was used.
19
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TABLE 5. EFFECT OF RABBIT ANTISERUM* ON KEPONE INHIBITION
OF DOG BRAIN (NEP) ATPASE ACTIVITIES WITH
SUBSTRATE ADDITION LAST
ATPase Activities
Additions Na+-K+ Mito Oligo
Enz+ATP .126 .065 .108
Control Ig (1. 2mg) +Enz (10' )-»-ATP .112 .074 .104
Ig IV (1.2mg)+Enz(10')-»-ATP .119 .066 .092
Kepone (3.75uM)+Enz(10')+ATP .102 .018 .087
Kepone (3 . 75yM) +Enz (5 ' ) ^-Control Ig(5' )->ATP .098 .033 .098
Kepone (3. 75yM) +Enz (5 ' ) -Ktg IV(5')+ATP .120 .068 .089
*Antiserum was fractionated and Ig IV was used.
The results in Table 4 show that nearly complete reversal
of inhibition by DCPD occurred for Na+-K+ATPase activity and
that some but much less reversal of mitochondrial Mg2+ATPase
activity was observed. However, apparently complete protection
was obtained against inhibition of ATPase activities by Kepone
in a dog brain preparation (Table 5).
With apparent success in the production of a specific anti-
body to Kepone and DCPD additional synthesis of Kelevan deriva-
tives of BSA, BGG and BF were carried out. As mentioned in the
Chemistry Studies, these synthetic reactions were conducted in
small batches because a poorer yield of derivative was obtained
when large batch synthesis was attempted. Also because of the
availability of chicken rearing facilities and the fact that
chickens produce larger quantities of immunoglobulins, fifteen
chickens were injected with BSA-Kel in complete Freund's
adjuvant. As mentioned earlier, the chickens responded with a
good production of antiserum as determined by micro-Ouchterlony
test and affinity column isolation (Figure 3). Reversal of
Kepone inhibition of ATPase activity by chicken produced antibody
to Kepone was observed from dog brain (Table 5), mice heart
(Table 6), and fly homogenate preparations (see last section of
report).
20
-------
TABLE 6. REVERSAL OF KEPONE INHIBITION OF MICE HEART
MITOCHONDRIAL MG++ATPASE ACTIVITY BY A PURIFIED
ANTIBODY FROM CHICKEN
Additions
Enzyme
+ Kepone**
+ Control-GG (Crude)
+ Control-GG (Crude) + Kepone
+ Ab (Crude)
+ Ab (crude) + Kepone
+ Control-GG (Purified)
+ Control-GG (Purified) + Kepone
+ Ab (Purified)
+ Ab (Purified) + Kepone
+ Control-GG (Purified)
+ Control-GG (Purified) + Kepone
+ Ab (Purified)
+ Ab (Purified) + Kepone
Antibody
--
2mg
2mg
2mg
2mg
62ug
62ug
62ug
62ug
124ug
124ug
124ug
124ug
ATPase
Activities*
10.97
5.98 r
10.47
5.90
10.07
10.10
10.50
5.70
10.47
8.98
10.40
5.90
10.70
9.98
Inhibition
%
--
0
46
0
0
0
46
0
18
0
46
0
9
Values are averages of 3 observations.
GG, y-globulin; Ab, Antibody.
* ymoles Pi rag~l protein hr~l.
**Kepone Cone. 3 x IQ-^M.
Isolation of Pure Antibody by Affinity Chromatography
The Sepharose-BGG-Kel preparation (see Materials and
Methods) was used to prepare a small affinity chromatography
column (0.6 x 11 cm). Small samples (0.4 ml) of control serum
and antiserum were tested for binding of Kepone reactive immuno-
globulins. Figure 3 shows typical results obtained from a number
21
-------
o
09
*-Ab.
•—CONT.
(2M KCI)
10
TUBE NO.
Figure 3. Elution patterns for control serum and BSA-Kelevan
antiserum from chickens. Blood samples allowed to clot and
sera added directly to a washed (Tris-Hcl, pH 7.0) BGG-Kel
Sepharose 4B affinity column. Large peak (left) unbound
protein. Peaks on right (0-control serum, *-antiserum, Ab.)
eluted with 2M KCI in Tris-HCl, pH 7.0.
22
-------
of such separations. Flow rate and fraction collection were set
to collect 2 ml per tube per 5 min. The large peak (left curve
Fig. 1) was the same for both blood serum samples (t-Control
serum, *-antiserum) and represents unbound protein that was
eluted with Tris-HCl buffer. The control serum samples always
contained a small amount of protein that bound to the affinity
column. However, the serum samples from BSA-Kel treated
chickens which contained "active antiserum yielded much larger
280 nm absorption peaks." It was observed that the above small
affinity column could determine the presence of antiserum for
Kepone in about one hour. By comparison the micro-Ouchterlony
test takes several days to indicate the presence of specific
antibodies to BGG-Kel.
The larger affinity column (see Materials and Methods) was
used to obtain sufficient quantity of the specific binding
protein (antibodies to the hapten Kelevan in BSA-Kel) to deter-
mine its effect on Kepone inhibition of the oligomycin (mito-
chondrial) sensitive Mg2+ ATPase activity from a mice heart
homogenate 13,000 x g fraction. (It was shown in other studies
by Anwar Saad in the P.I.'s laboratory that the Mg2+ATPase
activity in mice heart preparations was particularly sensitive
to Kepone.) Table 6 shows that BGG-Kel affinity column strong
protection against Kepone inhibition of ATPase activity was
observed at 124 yg protein per ml reaction mixture. Additional
specific antibody preparations from the affinity column showed
complete reversal of Kepone inhibition at less than 100 yg
protein per ml reaction mixture (see last section).
A sample of the specific binding protein from the affinity
column was analyzed by Dr. E. A. Lewis, Department of Chemistry,
University of Alabama, Tuscaloosa, Alabama, by ultracentrifuga-
tion. The results of his analysis are shown in Figure 4. Two
samples were analyzed: Sample A - the serum was fractionated by
Na2S04 and the partially purified Ig was passed through the
affinity column; Sample B - the blood serum was applied directly
to the affinity column. Dr. Lewis stated that there was no real
difference in sedimentation coefficients (S) for A and B. Sample
A was run at a higher concentration and probably accounts for
the small increase in S. The boundary profiles for both samples
were symmetrical and the samples must be homogeneous. Onlw one
late time (typical) cell boundary region tracing is shown i .
Figure 4. The sedimentation coefficient (S = 5.79 ave.) is
indicative of an immunoglobulin.
Effect of Antibody to Hapten (Kelevan) on Inhibition of ATPase
Activities by Pesticides Inhibitory to the ATPase System
A study was conducted after the termination date of this
grant (February 14, 1978) on the specificity of the antibody
produced against the antigen BSA-Kel. A sufficient quantity of
23
-------
0.82--
0.80- •
O
o>
o
0.78--
0.76
30 60 90
Time(min.)
120
150
A
c
0
u
C
O
u
Boundary Profile
T = 140 min.
Sedimentation
Figure 4. Ultracentrifugation analysis of specific binding protein
(Kepone antibody) from BGG-Kel affinity column. Slope A and B =
sedimentation coefficients. Boundary profile obtained bv
computer analysis of a series of boundary region tracings recorded
over approximately 2.5 hr
24
-------
purified antibody was not available prior to the termination date
to conduct these comparison studies. The synthesis of the BGG-
Kel affinity column and of good antibody production in 11 to 12
chickens in the last group of 15 chickens injected with BSA-Kel
made it possible to conduct these studies.
The results of preliminary experiments were surprising and
quite unexpected. It appears from the results that the antibody
to Kepone was generally effective against organochlorine pesti-
cides that inhibit the ATPase system. It was as effective in
preventing and reversing in vitro inhibition of mitochondrial
Mg2+ATPase activity from rat heart by Kepone (1 yM) and DDT
(2 yM). Inhibition of ATPase was also greatly reduced or
prevented by the antibody (72 yg/ml reaction mixture) using
reduced mirex (mixture of mono- and dihydro-winged derivatives)
(5 yM), Kelthane (2 yM), and aldrin (20 yM). In the experiments
with Kepone and DDT, reversal of ATPase inhibition was propor-
tional to amount of antibody added. As little as 7.2 yg anti-
body protein in 1 ml reaction mixture reduced the inhibition by
1 yM Kepone and 2 yM DDT by nearly 40%. Inhibition by Kepone
was completely prevented by 144 yg antibody. Inhibition by DDT
was only 6% in the presence of 144 yg antibody.
The above results indicate that the antibody to Kepone
produced by the antigen BSA-Kelevan has a rather broad specifi-
city to the organochlorine portion of these pesticides. To
determine this possibility, the protection by antibody of mito.-
Mg2+ATPase activity by Plictran and pyrethrin was investigated,
The inhibition by Plictran was not affected by the presence of
72 yg antibody. However, the inhibition by pyrethrin appeared
to be somewhat (20%) less in the presence of the antibody (72yg),
but the protection was not nearly as great at that observed for
the organochlorine pesticides. The latter observation with
pyrethrin will require further study; however, it appears that
the antibody produced by BSA-Kel has a broad specificity for
organochlorine pesticides that inhibit the ATPase system.
Further investigation of this observation is being con-
ducted. Concentration response studies are underway to determine
binding affinity relationships between the purified antibody
and the different organochlorine pesticides. The above findings,
if proved correct, indicate that the binding site for the
different organochlorine pesticide inhibitors on the ATPase
complexes must be the same or very similar. If this should
prove to be so, the reason for the difference in effectiveness
of different organochlorine pesticides may be related to their
ability to penetrate the lipoprotein complex of the enzyme
system. Studies to prove this latter possibility will be greatly
assisted by the availability of the antibody produced under
partial support by this EPA Grant.
25
-------
T. N. Patil
D. Desaiah
R. B. Koch
Biochemistry Department
26
-------
REFERENCES
Anderson, G. W., Zimmerman, J. E. and Callaham, M. F. (1964)
The use of esters of N-hydroxysuccinimide in peptide
synthesis. J. Am. Chem. Soc. 86, 1839-1842.
Auernheimer, A. H. and Atchley, F. 0. (1962) Modifications of
micro agar precipitin test. Am. J. Clin. Path. 38, 548-
553.
Brewer, F. D., Click, B. and Vinson, S. B. (1972) An immuno-
logical investigations of the factors responsible for the
resistance of Heliothis zea and susceptibility of Heliothis
virenscens to the parasitoid Cradiochiles nigriceps. Comp.
Biochem. Physiol. 431B, 781-786.
Campbell, D. H., Garvey, J. S. , Cremer, N. E., and Sussdorf, 0. H.
(1970) Methods in Immunology. W. A. Benjamin, Inc.,
Reading, Massachusetts. 454 pp.
Centeno, E. R., Johnson, W. J., and Sehon, A. H. (1970) Anti-
bodies to two common pesticides, DDT and Malathion. Int.
Arch. Allergy Appl. Immunol. 37, 1-13.
Cheng, E. Y. and Cutkomp, L. C. (1978) The Sensitivity of
Mitochondrial Mg2+ATPase to DDT and Analogs at Different
Temperature. In Press. Pest. Biochem. Physiol.
Cuatrecasas, P., Wilchek, M. and Anfinsen, C. B. (1968) Selec-
tive Enzyme Purification of Affinity Chromatography. Proc.
Natl. Acad. Sci. USA 61, 636-643.
Desaiah, D., Ho, I. K., and Mehendale, H. M. (1977) Effects of
Kepone and Mirex on mitochondrial Mg2 + at ATPase activitiy
in rat liver. Toxic. Appl. Pharmac. 39.
Desaiah, D. and Koch, R. B. (1975) Inhibition of ATPase activity
in channel catfish by Kepone and its reduction product.
Bull. Environ. Contam. Toxicol. 13, 153-158.
Desaiah, D., Koch, R. B., Cutkomp, L. K. and Jarvinen, A. (1975)
DDT: effect of continuous exposure on ATPase activity in
fish, Pimephales promelas. Arch. Environ. Contam. Toxicol.
3, 132-141.
27
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Fritz, P. J. and Hamrich, M. E. (1966) Enzymatic analysis of
adenosine triphosphatase. Enzymologia 30, 57-64.
Click, Bruce (1976) Immune-biology Laboratory Manual. Mississippi
State Printing Office, Mississippi State, MS 45pp.
Goodman, M. and Campbell, D. H. (1953) Differences in antigenic
specificity of normal human adult fetal and sickle cell
anemic hemaglobin. Blood 8, 422.
Hass, G. J. and Guardia, E. J. (1968) Production of antibodies
against insecticide-protein conjugates. Proc. Soc. Exp.
Biol. Med. 129, 546-551.
Katz, D. H., Paul, W. E., Goidl, E. A., and Benacerraf, B. (1970)
Carrier function in anti-hapten immune responses. I.
Enhancement of primary and secondary anti-hapten antibody
responses by carrier preimmunization. J. Exp. Med. 132,
261-282.
Koch, R. B. (1969a) Fractionation of olfactory tissue homogen-
ates. Isolation of a.highly concentrated plasma membrane
fraction. J. Neurochem. 16, 145-157.
Koch, R. B. (1969b) Chlorinated hydrocarbon insecticides:
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16, 269-271.
Koch, R. B. (1969/70) Inhibition of animal tissue ATPase
activities by chlorinated hydrocarbon pesticides. Chem.
Biol. Interact. 1, 199-209.
Koch, R. B., Desaiah, D., Click, B., Subba Rao, D. S. V., and
Stinson, R. (1977) Antibody Reactivation of Kepone Inhibited
Brain ATPase Activities. Gen. Pharm. 8, 231-234.
Kulkarni, M. S. and Click, B. (1974) Identification of serum
proteins following different stages of sodium sulfate
precipitation of chicken serum. Comp. Biochem. Physiol.
481B, 471-473.
Langone, J. J. and Van Vunakis, H. (1975) Radioimmunoassay for
dieldrin and aldrin. Res. Commun. Chem. Path. Pharmac. 10,
163-171.
Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J.
(1951) Protein measurements with the Folin phenol reagent.
J. Biol. Chem. 193, 265-275.
28
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Paul, W. E., Katz, D. H., Goidl, and B. Benacerraf (1970)
Carrier function in anti-hapten immune responses II.
Specific properties of carrier cells capable of enhancing
anti-hapten antibody responses. J. Exp. Med. 132:
283-299.
Pullman, M. E., Penefsky, H. S., Datta, A., and Racker, E. (1960,
Partial resolution of the enzymes catalyzing oxidative
phosphorylation. J. Biol. Chem. 235, 3322-3329.
Williams, C. A. and Chess, M. W. (1971) Methods in immunology
and immunology III. Reactions of antibodies with soluble
antigens. Academic Press, New York, 515 pp.
Wolfe, H. R. (1942) Precipitin production in chickens. I.
Interfacial titers as affected by quantity of antigen
injection and aging of antisera. J. Immunology 44: 135.
Wolfe, H. R. , Mueller, A., Neess, J., and Tempelis, C. (1957)
Precipitin production in chickens XVI. The relationship
of age to antibody production. J. Immunology 79: 142.
Wolfe, H. R., Amin, A., Mueller, A. P, and Aronson, F. R. (1960)
The secondary response of chickens given a primary inocula-
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Arch. Allergy Appl. Immunology 17:106.
29
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing!
REPORT NO.
EPA-600/3-78-093
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Determination of the Site(s) of Action of Selected
Pesticides by an Enzymatic-Immunobiological
Approach
5. REPORT DATE
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
Robert B. Koch
May 1977
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Mississippi State University
Department of Biochemistry
Mississippi State, Ms. 39762
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
EPA Grant R803458
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
13. TYPE OF REPORT AND PERIOD COVERED
Feb 15. 1975 to Feb 14. 1978_
14. SPONSORING AGENCY CODE
EPA/600/4
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This report describes development of an antibody to an organochlorine pesticide
to be used in studies related to its inhibition of the ATPase system. Kelevan,
the condensation product of ethyl levulinate and Kepone, was successfully con-
jugated to bovine serum albumin (BSA), fibrinogen (BF) , and gamma globulin (BGG).
Rabbits and chickens preimmunized with BSA and then immunized with BSA-Kelevan
produced antibodies to both the hapten, Kelevan, and the carrier protein BSA.
Antiserum to Kelevan protected ATPase activity against Kepone and its derivatives.
The titer of antibody to Kelevan was critical since antiserum with only trace
amounts of Kelevan antibody failed to protect the ATPase activity against Kepone
Inhibition. Antibody was concentrated by Na2S04 fractional precipitation of the
antiserum and obtained in pure form by affinity chromatography using BGG-Kel
covalently linked to Sepharose 4B. Pure antibody was obtained from untreated blooc
serum or plasma with no prior pretreatment or fractionation using the BGG-Kel
affinity column. Complete protection of mitochondrial mg^+ATPase activity from
in vitro inhibition of Kepone was obtained using a 1.2 mg quantity of naoSO/^
fractionated antibody arid only 120 yg of pure antibody. Reversal of ATPase
inhibition was readily obtained by addition of antibody prior to addit n ,?f
substrate to the reaction mixture.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Enzymes
Immune serums
Immunization
Pesticides
b.IDENTIFIERS/OPEN ENDED TERMS
Antibodies
Organochlorine Pesticides
Kelevan
Kepone
albrimin
fibrinogen
gamma globilin
COSATI I icki litvup
6F
6A
6E
18. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS I Hiis Report I
Uncla s..s. 1 f ie d
20 SECURITY CLASS i This pa/to
21. NO OF PAGES
29
22. PRICE
EPA Form 2220-1 (Rev. 4-77) previous i r>i TION is OPSOLK TF.
°"8-6-OOV1700. Region 4.
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